Improved recombinant polyadenylation signal sequences and uses thereof

Abstract

The present invention relates to improved recombinant polyadenylation signal sequences and uses thereof.

Description

Technical Field

[0001] This invention relates to an improved recombinant polyadenylation signal sequence and its uses. Background Technology

[0002] Developing stable, high-level recombinant protein expression cell lines is crucial for cell engineering applications in research, disease modeling, drug discovery, therapeutic gene expression, and biopharmaceutical manufacturing. Achieving robust recombinant protein expression in desired host cells depends on optimizing expression vectors using various enhancers, promoters, introns, polyA, and regulatory sequences. However, while various approaches have been reported for developing and optimizing genetic elements used in expression vectors to achieve desired levels of transgenic expression, including assembling naturally occurring or newly designed blocks of functional elements (Cao et al., 2021; McFarland et al., 2006; Patel et al., 2021; Schlabach et al., 2010), few have considered creating higher-level multi-gene expression vectors to predictably co-express recombinant genes from the same vector. Because most efforts to optimize expression vectors focus on identifying the optimal sequence for a specific recombinant protein and vector, the translatability of these genetic elements to other applications may be limited. Furthermore, using an optimized genetic element and replicating it for multiple expression units on a multigene expression vector is generally not ideal, because the replication of the sequence by regulating the replication or the introduction of extended regions of repetitive sequences by reusing the same genetic element introduces the risk of recombination events 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 crucial for efficient protein expression of endogenous genes and recombinant genes expressed from engineered vectors. Specifically, the addition of a poly-A tail to protein-coding transcripts contributes to nuclear export and translation, as well as mRNA stability, by protecting the transcripts from enzymatic degradation in the cytoplasm. Some of the more commonly used polyadenylation signal sequences used in vector development include sequences from bovine growth hormone (BGH) (Goodwin et al., 1992), human growth hormone (hGH) (Pfarr et al., 1986), simian virus 40 (SV40) (Hans et al., 2000), and rabbit β-globin (RbG) (Lanoix et al., 1988). However, the choice of polyadenylation signal sequence can significantly influence the properties of expression vectors due to significant differences in size and sequence composition. Larger sizes can contribute to reduced transfection and integration efficiency during engineered cells and, in the case of viral vector engineering, may increase the size of the genetic load beyond the viral vector's packaging capacity. Differences in sequence composition and functional elements within polyadenylation signaling sequences can lead to heterogeneous expression levels due to their respective uses and differences in expression levels between different host cells. For example, in the cases of SV40 and RbG, these sequences are more efficient polyadenylation signaling sequences compared to others due to the presence of additional upstream and downstream functional elements that facilitate termination and polyadenylation (Gil et al., 1987; Schek et al., 1992). Furthermore, recent data suggest that even small variations in sequence composition can lead to differences in transcriptional termination processes and protein expression within the same host cell and between various host cells (Omelina et al., 2022).

[0004] Therefore, improvements to the polyadenylation signaling sequence are still needed. Summary of the Invention

[0005] This paper provides a library of robust transcription termination and polyadenylation (polyA) signal sequences for advanced vector development. The inventors have generated a set of rationally designed recombinant polyadenylation signal sequences based on a minimal core sequence of the RbG polyadenylation signal sequence (Levitt et al., 1989). These recombinant polyadenylation signal sequences are particularly useful for generating multigene expression vectors for engineered mammalian cells due to their small size and defined functional element composition. Furthermore, the sequence composition has been specifically designed to adapt to Gibson DNA assembly cloning to facilitate the generation of multigene expression vectors and has low sequence identity (sequence identity) to reduce the risk of DNA recombination events. The resulting recombinant polyadenylation signal sequences of this invention support significantly higher expression levels than known short polyadenylation signal sequences (such as core RbG polyA sequences) (see, for example, Figure 5) and levels comparable to larger, commonly used polyadenylation signal sequences (see, for example, Figure 6). Furthermore, the inventors demonstrate that the recombinant polyadenylation signaling sequences of the present invention are minimally affected by the composition of the upstream 3'UTR sequence (see, for example, Figure 7) and can support robust expression with constitutive promoter combinations of varying strengths (see, for example, Figure 8). In summary, the inventors provide a novel and improved set of recombinant polyadenylation signaling sequences that can facilitate the development of advanced multigene expression vectors and cell models for research, disease modeling, drug discovery, therapeutic gene expression, and biopharmaceutical manufacturing. Further advantageous effects in the context of specific exemplary uses are described below.

[0006] In one embodiment, a recombinant transcription unit comprising a nucleotide sequence encoding a polypeptide is provided, wherein the nucleotide sequence is operatively linked to a recombinant polyadenylation signal sequence, wherein the recombinant polyadenylation signal sequence has a sequence length of less than 100 nucleotides, and wherein 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 level as the polypeptide in eukaryotic cells transformed with a reference nucleic acid comprising the recombinant polyadenylation signal sequence consisting 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 sequence identity.

[0007] In some embodiments, 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 the nucleotide 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.

[0008] In some embodiments, a recombinant transcription unit comprising a nucleotide sequence encoding a polypeptide is provided, wherein the nucleotide sequence is operatively linked to a recombinant polyadenylation signal sequence, wherein the recombinant polyadenylation signal sequence comprises or is composed of a nucleotide sequence selected from or consisting of 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.

[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 recombinant nucleic acid is provided, comprising:

[0011] (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

[0012] (b) A second recombinant transcription unit comprising a second nucleotide sequence encoding a second polypeptide operably linked to a second recombinant polyadenylation signal sequence.

[0013] The first recombinant polyadenylation signal sequence and the second recombinant polyadenylation signal sequence have less than 70%, 65%, 60%, 55%, 50%, 45%, or 40% sequence identity.

[0014] In some embodiments, the recombinant nucleic acid further comprises:

[0015] (c) A third recombinant transcription unit comprising a third nucleotide sequence encoding a third polypeptide that is operatively linked to a third recombinant polyadenylation signal sequence.

[0016] The first and second recombinant polyadenylation signal sequences individually share less than 70%, 65%, 60%, 55%, 50%, 45%, or 40% sequence identity with the third recombinant polyadenylation signal sequence.

[0017] In some embodiments, 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.

[0018] In some embodiments, the first recombinant polyadenylation signal sequence, the second recombinant polyadenylation signal sequence, and, if present, the third recombinant polyadenylation signal sequence, cannot participate in DNA strand exchange to form a recombinant intermediate.

[0019] 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.

[0020] 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.

[0021] In some embodiments, the first polypeptide, the second polypeptide, and, if present, the third polypeptide are expressed in eukaryotic cells.

[0022] In some embodiments, the first recombinant transcription unit is a recombinant transcription unit as described above, and the second recombinant transcription unit is a recombinant transcription unit as described above, and the third recombinant transcription unit, if present, is a recombinant transcription unit as described above.

[0023] In some embodiments, a recombinant nucleic acid as described above is provided, wherein

[0024] (a) The first recombinant transcription unit further comprises a first promoter operatively linked to a nucleotide sequence encoding a first polypeptide, and

[0025] (b) The second recombinant transcription unit further comprises a second promoter operatively linked to a nucleotide sequence encoding a second polypeptide.

[0026] The first promoter and the second promoter have sequence identity of less than 70%, 65%, 60%, 55%, 50%, 45%, or 40%.

[0027] In some embodiments, a recombinant nucleic acid as described above is provided, which further includes:

[0028] (c) If the first recombinant transcription unit further comprises a first promoter operatively linked to a nucleotide sequence encoding a first polypeptide,

[0029] The first and second promoters have sequence identity with the third promoter of less than 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 65%, or 60%.

[0030] In some embodiments, a recombinant nucleic acid as described above is provided, wherein

[0031] (i) The first promoter, the second promoter, and, if present, the third promoter, are active in eukaryotic cells.

[0032] (ii) The first promoter drives the expression of the first polypeptide.

[0033] (iii) The second promoter drives the expression of the second polypeptide.

[0034] (iv) The third promoter drives the expression of the third polypeptide, and / or

[0035] (v) The first promoter, the second promoter, and, if present, the third promoter drive the expression of the first polypeptide, the second polypeptide, and, if present, the third polypeptide, respectively.

[0036] In some embodiments, a recombinant nucleic acid as described above is provided, wherein the first promoter, the second promoter, and, if present, the third promoter are individually selected from the group consisting of the hPGK1 promoter, the CMV promoter, and the hEF1α promoter.

[0037] In some embodiments, the recombinant nucleic acid comprises at least one vector.

[0038] In some embodiments, the recombinant nucleic acid comprises: a first vector containing a first recombinant transcription unit; a second vector containing a second recombinant transcription unit; and a third vector containing a third recombinant transcription unit if a third recombinant transcription unit is present.

[0039] In some embodiments, at least one vector comprises an optional marker operatively linked to a first recombinant transcription unit, a second recombinant transcription unit, or, if present, a third recombinant transcription unit.

[0040] In some embodiments, the selectable markers are selected from the group consisting of: hygromycin selectable markers, neomycin selectable markers, G418 selectable markers, dihydrofolate reductase (DHFR), thymidine kinase, glutamine synthase, asparagine synthase, tryptophan synthase, histidine dehydrogenase, and nucleic acids conferring resistance to puromycin, bleomycin, fulvicin, chloramphenicol, bleomycin, and mycophenolic acid.

[0041] In some embodiments, the first vector, the second vector, and / or, if present, the third vector, contains a bacterial origin of replication, particularly the pUC19 origin of replication.

[0042] In some embodiments, a host cell is provided comprising the recombinant transcription unit as described above and / or the recombinant nucleic acid as described above.

[0043] In some embodiments, the host cell is a eukaryotic host cell.

[0044] In some embodiments, the host cell is selected from the group consisting of CHO, BHK, HEK and Sp2 / O.

[0045] In some embodiments, a recombinant viral vector comprising a vector genome is provided, wherein the vector genome is comprised in a 5' to 3' sequence:

[0046] (i) 5' ITR sequence,

[0047] (ii) Startup subsequence

[0048] (iii) The sequence encoding the polypeptide.

[0049] (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

[0050] (v)3' ITR sequence.

[0051] In some embodiments, 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.

[0052] In some embodiments, the recombinant viral vector is selected from the group consisting of retroviral vectors, adenovirus vectors, helper-dependent adenovirus vectors, heterozygous adenovirus vectors, herpes simplex virus vectors, lentiviral vectors, poxvirus vectors, Epstein-Barr virus vectors, vaccinia virus vectors, human cytomegalovirus vectors, lentiviral vectors, adenovirus vectors or adeno-associated virus (AAV) vectors, or recombinant variants derived therefrom.

[0053] In some embodiments, the recombinant viral vector is a recombinant adeno-associated virus (rAAV) vector.

[0054] 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 variant capsids derived therefrom.

[0055] In some embodiments, a method for generating a target peptide is provided, the method comprising the following steps:

[0056] (a) Provide the host cells as described above,

[0057] (b) Incubate host cells under conditions suitable for peptide expression.

[0058] (c) Recover the target peptide from the cell culture.

[0059] In some embodiments, a method for generating a target peptide is provided, the method comprising the following steps:

[0060] (a) Providing a host cell comprising the recombinant nucleic acid as described above, wherein the target polypeptide is a first polypeptide, and wherein a second polypeptide and, if present, a third polypeptide are necessary for or improve the production of the target polypeptide.

[0061] (b) Incubate host cells under conditions suitable for the expression of the first polypeptide, the second polypeptide, and, if present, a third polypeptide.

[0062] (c) Recovery of the target peptide from cell cultures, and optionally

[0063] (d) Formulate the recovered target peptide for therapeutic use.

[0064] In some embodiments, a method for generating a recombinant adeno-associated virus (rAAV) vector is provided, the method comprising the following steps:

[0065] (a) Providing a host cell containing the recombinant nucleic acid as described above, wherein a first polynucleotide sequence encodes a therapeutic payload, wherein a second nucleotide sequence encodes the viral vector rep and cap proteins, and wherein a third nucleotide sequence encodes the E4, E2a, and VA proteins.

[0066] (b) Incubate host cells under conditions suitable for generating the recombinant rAAV vector, and

[0067] (c) Recovery of viral vectors from cell cultures, and optionally

[0068] (d) Formulate the recovered target peptide for therapeutic use.

[0069] In some embodiments, the method described above is provided, wherein the host cell is selected from the group consisting of CHO cells, BHK cells, HEK cells, and Sp2 / O cells.

[0070] In some embodiments, the method described above is provided, 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.

[0071] In some embodiments, a recombinant transcription unit is provided for recombination to generate a target polypeptide, wherein the recombinant transcription unit is as defined above.

[0072] In some embodiments, the recombinant nucleic acid is provided for recombination to generate a target polypeptide, wherein the recombinant nucleic acid is as defined above. Attached Figure Description

[0073] Figure 1 is a schematic diagram of the reporter plasmid used to test the recombinant polyadenylation signal sequence, which consists of a constitutive promoter (Prom.), enhanced green fluorescent protein (EGFP), P2A self-cleaving peptide sequence, NanoLuc luciferase (Nluc), PEST protein degradation signal and 3' untranslated region (3'UTR), and the target recombinant polyadenylation signal sequence is downstream of it.

[0074] Figure 2 (2A) Schematic diagram of reporter plasmids with BGH or 2x sNRP-1 polyadenylation signal sequences. (2B) Transient detection of the corresponding reporter constructs in HEK293T at Nluc expression levels assessed 24 hours post-transfection. Bars represent the mean ± standard deviation of the mean relative Nluc luminescence (RLU) (normalized luminescence; %) of n = 16 biologically independent samples normalized to BGH polyA-encoded reporter plasmids.

[0075] Figure 3. Schematic diagram of the 95 nt recombinant polyadenylation signal sequence design provided in this paper. The recombinant polyadenylation signal sequence design includes (i) a 46 nt U-rich heterologous upstream sequence element (USE) region designed to contain a unique primer annealing site with a Tm of 70-72°C, compatible with Gibson Assembly; (ii) a polyadenylation signal (PAS); (iii) a variable spacer region containing two cytosine-adenosine monophosphate (CA) mRNA cleavage sites located 15-20 nt downstream of the PAS; and (iv) two GU / U-rich downstream sequence elements (DSE; DSE1 and DSE2) regions.

[0076] Figure 4 (4A) Schematic diagram of the reporter plasmid containing the novel recombinant polyadenylation signal sequence. (4B) Transient detection of the corresponding reporter construct in HEK293T at Nluc expression levels assessed 24 hours post-transfection. Bars represent the mean ± standard deviation of n = 16 independent replicates of the normalized mean Nluc relative luminescence (RLU) (normalized luminescence; %) across all samples.

[0077] Figure 5 (5A) Schematic diagram of a reporter plasmid containing one of the rabbit β-globulin polyadenylation signal sequences defined by Levitt et al. or one of three selected recombinant polyadenylation signal sequences (polyA-2.3, -3.3, -4.3). (5B) Transient detection of the corresponding reporter construct in HEK293T at Nluc expression levels assessed 24 hours post-transfection. Bars represent the mean ± standard deviation of the mean relative Nluc luminescence (RLU) (normalized luminescence; %) of n = 16 independent replicates normalized to the polyA-encoded reporter plasmid samples by Levitt et al.

[0078] Figure 6 (6A) Schematic diagram of reporter plasmids containing one of hGH, BGH, SV40, or three selected recombinant polyadenylation signal sequences (polyA-2.3, -3.3, -4.3). (6B) Transient detection of the corresponding reporter construct in HEK293T at Nluc expression levels assessed 24 hours post-transfection. Bars represent the mean ± standard deviation of the mean Nluc relative luminescence (RLU) (normalized luminescence; %) of n = 16 independent replicates normalized to the BGH polyA-encoded reporter plasmid sample.

[0079] Figure 7 (7A) Schematic diagram of a reporter plasmid containing one of three selected recombinant polyadenylation signal sequences (polyA-2.3, -3.3, -4.3) combined with three different de novo designed 3'UTR sequences (3'UTR 1, 2, 3). (7B) Transient detection of the corresponding reporter construct in HEK293T at Nluc expression levels assessed 24 hours post-transfection. Bars represent the mean ± standard deviation of n = 16 independent replicates of the normalized mean Nluc relative luminescence (RLU) (normalized luminescence; %) for all samples.

[0080] Figure 8 (8A) shows a schematic diagram of a reporter plasmid containing one of three selected recombinant polyadenylation signal sequences (polyA-2.3, -3.3, -4.3) combined with three constitutive promoters (hPGK1, CMV, hEF1α) of varying intensities. (8B-8D) Transient detection of reporter constructs driven by (8B) hPGK1-, (8C) CMV-, and (8D) hEF1α in HEK293T, evaluated at Nluc expression levels 24 hours post-transfection. Bars represent the mean ± standard deviation of n = 16 independent replicates of normalized average Nluc relative luminescence (RLU) (normalized luminescence; %) for all samples tested with the corresponding promoter.

[0081] References

[0082] Bzymek et al. (2001). Instability of repetitive DNA sequences: the role of replication in multiple mechanisms. Proceedings of the National Academy ofSciences, 98(15), 8319-8325.

[0083] Batt et al. (1995). Characterization of the polyomavirus late polyadenylation signal. Molecular and Cellular Biology, 15:4783-4790

[0084] Cao et al. (2021). High-throughput 5′ UTR engineering for enhanced protein production in non-viral gene therapies. Nature communications, 12(1), 4138.

[0085] Cole et al. (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

[0086] Finn et al. (1989). Homologous plasmid recombination is elevated in immortalized transformed cells. Molecular and Cellular biology, 9(9), 4009-4017.

[0087] Gil et al. (1987). Position-dependent sequence elements downstream of AAUAAA are required for efficient rabbit β-globin mRNA 3′ end formation. Cell, 49(3), 399-406.

[0088] Gil et al. (1984). A sequence downstream of AAUAAA is required for rabbit β-globin mRNA 3′-end formation. Nature, 312: 473-474

[0089] Gimmi et al. (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

[0090] Goodwin et al. (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.

[0091] Hans et al. (2000). Functionally significant secondary structure of the simian virus 40 late polyadenylation signal. Molecular and Cellular Biology, 20(8), 2926-2932.

[0092] Lanoix et al. (1988). A rabbit beta‐globin polyadenylation signal directs efficient termination of transcription of polyomavirus DNA. The EMBO journal, 7(8), 2515-2522.

[0093] Levitt et al. (1989). Definition of an efficient synthetic poly (A) site. Genes & Development, 3(7), 1019 - 1025.

[0094] McFarland et al. (2006). Evaluation of a novel short polyadenylation signal as an alternative to the SV40 polyadenylation signal. Plasmid, 56(1), 62 - 67.

[0095] Murthy et al. (1995). The 160 - kD subunit of human cleavage - polyadenylation specificity factor coordinates pre - mRNA 3'-end formation. Genes & Development 9:2672 - 2683

[0096] Omelina et al. (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

[0097] Patel et al. (2021). Control of multigene expression stoichiometry in mammalian cells using synthetic promoters. ACS Synthetic Biology, 10(5), 1155 - 1165

[0098] Pfarr et al. (1986). Differential effects of polyadenylation regions on gene expression in mammalian cells. DNA, 5(2), 115-122

[0099] Schek et al. (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

[0100] Schlabach et al. (2010). Synthetic design of strong promoters. Proceedings of the National Academy of Sciences, 107(6), 2538-2543

[0101] Takagaki et al. (1997). RNA recognition by the human polyadenylation factor CstF. Molecular and Cellular Biology 17: 3907–3914

[0102] 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 Detailed implementation manners

[0103] The inventors have generated improved polyadenylation signal sequences that, when integrated into a transcription unit (also known as a transcription cassette), lead to strong expression of the target polypeptide. The new sequences are short, which is advantageous for many applications, such as integrating recombinant polyadenylation signal sequences into transcription units of limited size (e.g., in the context of recombinant adeno-associated virus vectors). Furthermore, the inventors have generated multiple recombinant polyadenylation signal sequences sharing low sequence homology, i.e., sequence identity among the multiple sequences thus preventing recombination events. Recombination events may occur in eukaryotic cells if sequences with high sequence homology are close to each other, for example, if sequences with high sequence homology are integrated into the same genomic locus, or if multiple plasmid vectors sharing high homology sequences are transfected into cells.

[0104] As illustrated by examples, short polyadenylation signal sequences known in the art (such as the 2x sNRP-1 signal sequence described by McFarland et al., SEQ ID NO:14, 49 nucleotides long) do not affect the expression level of the target peptide in a manner comparable to that of longer polyadenylation signal sequences known in the art (such as the BGH sequence, SEQ ID NO:16, 208 nucleotides long). This is illustrated, for example, in Figure 2B.

[0105] In contrast, the novel recombinant polyadenylation signal sequences of the present invention (SEQ ID NO 1-12) exhibit similar or higher strong expression levels compared to longer polyadenylation signal sequences known in the art (such as, for example, hGH, BGH, and SV40 sequences, SEQ ID NO: 15-17, 122-477 nucleotides long). For example, in Figures 4B, 5B, and... Figure 6B As shown in the image.

[0106] Therefore, this document provides novel and improved recombinant polyadenylation signal sequences. Further, recombinant transcription units comprising the improved recombinant polyadenylation signal sequence according to the invention are provided. These transcription units can influence the strong expression of nucleotide sequences encoding target polypeptides and are operatively linked to the improved recombinant polyadenylation signal sequence according to the invention.

[0107] In one aspect, a recombinant nucleic acid comprising a recombinant transcription unit containing a nucleotide sequence encoding a polypeptide, wherein the nucleotide sequence is operatively linked to a recombinant polyadenylation signal sequence.

[0108] The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” are used interchangeably to refer to multiple “nucleotides” (i.e., molecules containing a sugar (e.g., ribose or deoxyribose) linked to a phosphate group and an exchangeable organic base, which is a substituted pyrimidine (e.g., cytosine (C), thymine (T), or uracil (U)) or a substituted purine (e.g., adenine (A) or guanine (G)). As used herein, the term refers to oligonucleotides as well as oligodeoxynucleotides. These terms should also include polynucleotides (i.e., polynucleotides minus phosphate) and any other organic base-containing polymers. Nucleic acid molecules can be obtained from existing nucleic acid sources (e.g., genomes or cDNA) but can also be synthesized (e.g., through oligonucleotide synthesis).

[0109] As mentioned in this article, “recombinant” nucleic acids refer to nucleic acids that are not naturally occurring. Recombinant nucleic acids can also be called “synthetic” nucleic acids. Similarly, recombinant transcription units and recombinant polyadenylation signal sequences refer to non-naturally occurring transcription units and polyadenylation signal sequences, such as those containing or composed of recombinant nucleic acids. Recombinant nucleic acids may contain or be composed of polynucleotide sequences that contain and / or encode the genome of a non-naturally occurring organism (e.g., a wild-type organism). Recombinant nucleic acids may contain or be composed of polynucleotide sequences that are not included in the polynucleotide sequences of (RNA) transcripts produced by naturally occurring organisms. Recombinant nucleic acid technologies can be used to produce recombinant nucleic acids. Recombinant nucleic acid technologies include techniques for constructing and manipulating nucleotide sequences of nucleic acids and include molecular cloning.

[0110] The term "transcription unit" refers to a DNA sequence that encodes a single RNA molecule, such as an mRNA molecule. A transcription unit includes the nucleotide sequence necessary for transcription; for example, a transcription unit typically includes a promoter, a multinucleotide sequence encoding the target protein, and a terminator sequence (such as the 3' untranslated region, also known as the 3'-UTR).

[0111] The term "operably linked" refers to a situation where the nucleic acid encoding the target recombinant polypeptide and a regulatory nucleic acid sequence (e.g., a polyadenylation signal, promoter, and / or enhancer) are covalently linked in such a way that the expression of the nucleic acid encoding the target polypeptide is placed under the influence or control of the regulatory nucleic acid sequence (thus forming a transcription unit or expression cassette). Therefore, if the (regulatory) sequence can influence the transcription of a selected nucleic acid sequence, then the regulatory sequence is operably linked to that nucleic acid sequence. The resulting transcript can then be translated into the desired target polypeptide.

[0112] The term "polyadenylation signal sequence" refers to a sequence that terminates transcription of a transcription unit and ensures that the nucleic acid sequence encoding a polypeptide is correctly transcribed and translated. The polyadenylation signal is recognized by the RNA cleavage complex, leading to RNA cleavage and polyadenylation catalyzed by polyadenylate polymerase.

[0113] Examples of naturally occurring eukaryotic polyadenylation signals include the rabbit β-globin poly(A) signal, which has been characterized as strong in the literature (Gil and Proudfoot, Cell 49: 399-406 (1987); Gil and Proudfoot, Nature 312: 473-474 (1984)). One of its key features is the structure of its downstream components, which contain UG- and U-rich domains. Other polyadenylation signaling sequences include synthetic polyA, HSV thymidine kinase polyA (see Cole, CN and TP Stacy, MoI. Cell. Biol. 5:2104-2113 (1985)); human alpha globulin polyA SV40 polyA (see Schek, N, Cooke, C and JC Alwine, MoI. CellBiol. 12:5386-5393 (1992)); human β globulin polyA (see Gil, A., and NJ Proudfoot, Cell 49:399-406 (1987)); polyomavirus polyA (see Batt, D. B and GG Carmichael MoI. Cell. Biol. 15:4783-4790 (1995); bovine growth hormone polyA (Gimmi, ER, Reff, ME, and IC Deckman, Nucleic A). Acid Res. (1989)).

[0114] Additional polyadenylation sites can be identified or constructed using methods known in the art. The smallest polyadenylation site consists of AAUAAA and a second recognition sequence (typically a G / U-rich sequence), with approximately 30 nucleotides downstream. As used herein, the sequence is presented as DNA, not RNA, to facilitate the preparation of suitable DNA for incorporation into expression vectors. When presented as DNA, the polyadenylation site consists of AATAAA, for example, with 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 polyadenylation-specific factor (CPSF; Murthy KG and Manley JL (1995), Genes Dev 9:2672-2683), and the second site (typically 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 responsible for RNA binding is CstF-64, which is a member of the ribonucleoprotein domain protein family (Takagaki et al. (1992) Proc Natl Acad Sci USA 89:1403-1407).

[0115] Without being bound by theory, polyadenylation signaling sequences should be understood as 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. 3' regulatory elements are not translated into proteins but play important roles in regulating gene expression, such as influencing the stability, localization, and translation of mRNA transcripts, and ultimately affecting the expression (level) of protein-coding genes.

[0116] This document provides an improved recombinant polyadenylation signal sequence with several enhanced properties. The recombinant polyadenylation signal sequence of this invention results 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 sequence of this invention is shorter than effective polyadenylation signal sequences known in the art, for example, compared to polyadenylation signal sequences selected from rabbit β-globin poly(A) signal, HSV thymidine kinase poly(A), human α-globin poly(A), SV40 poly(A), human β-globin poly(A), polyomavirus poly(A), and bovine growth hormone poly(A).

[0117] In one embodiment, the recombinant polyadenylation signal sequence disclosed herein comprises less than 100, 99, 98, 97, or 96 nucleotides. In one embodiment, the recombinant polyadenylation signal sequence disclosed herein has a sequence length of less than 100, 99, 98, 97, or 96 nucleotides. In one embodiment, the recombinant polyadenylation signal sequence disclosed herein has a sequence length between 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 aspect, the recombinant polyadenylation signal sequence disclosed herein is recognized by an RNA polymerase, thereby causing the RNA polymerase to release RNA molecules. In one aspect, the RNA polymerase is a eukaryotic RNA polymerase. In one aspect, a eukaryotic cell containing a transcription unit is capable of expressing the polypeptide, the transcription unit comprising a nucleotide sequence encoding the polypeptide, wherein the nucleotide sequence is operatively linked to a recombinant polyadenylation signal sequence as disclosed herein. In one aspect, the recombinant polyadenylation signal sequence results in the (transcriptional) termination and polyadenylation of the mRNA transcript of the transcription unit in the eukaryotic cell. Thus, the recombinant polyadenylation signal sequence initiates transcriptional termination of the transcription unit.

[0118] Without theoretical constraints, short regulatory elements are advantageous in many applications of recombinant transcription units. For example, the size of viral vectors can be limited. Recombinant adeno-associated virus (rAAV) is limited to AAV transgenes smaller than 5 kilobases, and the transgene needs to include the coding sequence of the target gene as well as the promoter sequence, enhancer, and polyadenylation signal. Short regulatory elements leave more space for the coding sequence.

[0119] The term "expression" refers to the process of using information from nucleic acids to synthesize a functional polynucleotide that produces a (genetic) product, such as a target protein. Expression can include transcription, RNA splicing, translation, and post-translational modifications. Expression regulation can control the timing, location, and quantity of a given expression product (such as a target protein) present in the cell.

[0120] The term "termination" refers to the process by which RNA polymerase stops adding nucleotides to the growing RNA chain and releases the RNA molecule. "Polyadecylation" refers to the process of adding an adenine nucleotide chain (also known as a poly(A) tail) to the 3' end of a newly synthesized RNA molecule. Polyadecylation is catalyzed by poly(A) polymerase and occurs after the RNA molecule is cleaved at a specific site downstream of the coding region (polyadecylation signal). Termination and polyadecylation refer to two processes that consecutively produce mature mRNA transcripts.

[0121] "mRNA transcripts," also known as "messenger RNA" or simply "mRNA," are RNA molecules that carry genetic information from DNA in the cell nucleus to ribosomes, where they serve as templates for protein synthesis. During transcription, the DNA sequence of a protein-coding gene is used as a template to generate complementary RNA molecules, which are then processed and modified to form mature mRNA transcripts. Modifications that lead to mature mRNA transcripts include, for example, 5' capping, splicing, and polyadenylation.

[0122] In one embodiment, a recombinant transcription unit comprising a nucleotide sequence encoding a polypeptide is provided, wherein the nucleotide sequence is operatively 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 level as the polypeptide in eukaryotic cells transformed with a reference nucleic acid comprising the recombinant polyadenylation signal sequence consisting 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, regardless of sequence identity.

[0123] The term "expression level" refers to a quantitative determination of the level at which a cell expresses a specific open reading frame (such as that included in a transcription unit). Expression levels can be determined, for example, by detecting the product of the open reading frame (such as a protein) using methods known in the art, such as Western blot analysis. However, it is often easier to detect one of the protein's precursors, such as mRNA, and infer gene expression levels from these measurements. mRNA levels can be quantitatively measured using methods known in the art, such as, for example, Northern blotting, RT-qPCR, or hybridization microarrays. In one embodiment, expression levels are determined by RT-qPCR analysis. Another method for determining expression levels is by using a reporter gene (also called a reporter), which is a gene that can be readily identified and measured when operatively linked to a regulatory sequence, for example by fluorescence or luminescence. Such reporter genes are well known in the art and are also described herein.

[0124] For example, in Example 2, a recombinant transcription unit comprising the luminescent protein NanoLuc luciferase is described. The expression level of luciferase can be determined by methods known in the art and the method shown in Example 1.3.

[0125] In one embodiment, expression levels are influenced by a recombinant transcription unit comprising a nucleotide sequence encoding a polypeptide, wherein the nucleotide sequence is operatively linked to a recombinant polyadenylation signaling sequence as determined in the following manner:

[0126] (a) A reporter transcription unit is generated by providing the nucleotide sequence of a recombinant transcription unit and replacing the nucleotide sequence encoding a polypeptide with the nucleotide sequence encoding luciferase of SEQ ID NO:19.

[0127] (b) HEK293T cells were transfected with a reporter plasmid containing a reporter transcription unit, and HEK293T cells were cultured under conditions suitable for reporter transcription unit expression.

[0128] (c) Measure luciferase levels 24 hours after transfection.

[0129] In one embodiment, the expression level of the recombinant nucleic acid receiving a first expression level is determined as described above, and then the expression level of the reference nucleic acid receiving a second expression level is determined as described above, 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.

[0130] As used herein, a “reference nucleic acid” refers to a nucleic acid that is similar to or identical to a target recombinant nucleic acid (such as a recombinant nucleic acid containing the recombinant transcription unit of the present invention) except for the target sequence element. A reference nucleic acid can be used to compare or benchmark the functionality (e.g., affected expression level) of a target recombinant nucleic acid with a specific reference nucleic acid (e.g., a nucleic acid containing a recombinant polyadenylation signal sequence consisting of the nucleotide sequence of SEQ ID NO:2). In some aspects, the nucleotide sequence of the reference nucleic acid is identical to that of the target recombinant nucleic acid, except (without regard to sequence identity) the target sequence element, such as, for example, the recombinant polyadenylation signal sequence of the present invention. In some aspects, the nucleotide sequence of the recombinant polyadenylation signal sequence is not considered when determining sequence identity. For example, the nucleotide sequence of the recombinant polyadenylation signal sequence can be omitted (deleted) from both the target recombinant nucleic acid and the reference nucleic acid for sequence comparison.

[0131] In one embodiment, eukaryotic cells transformed with a recombinant nucleic acid containing a recombinant transcription unit (alone) are able to express the polypeptide at the same or higher expression level as the polypeptide in eukaryotic cells of the same type transformed with a reference nucleic acid containing a recombinant polyadenylation signal sequence consisting of SEQ ID NO:2, wherein the nucleotide sequence of the reference nucleic acid is identical to that of the recombinant nucleic acid, without regard to sequence identity. In one embodiment, HEK293T cells are transfected with a reporter plasmid containing the recombinant transcription unit (alone) by integrating each of the recombinant polyadenylation signal sequences (in 5'-3' sequence) into a recombinant transcription unit containing the nucleotide sequence of SEQ ID NO:19 (encoding NanoLuc luciferase) and the target recombinant polyadenylation signal sequence, HEK293T cells are cultured under conditions suitable for expressing the recombinant transcription unit, and the expression level is determined by measuring the luciferase level 24 hours post-transfection.

[0132] In one embodiment, 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 the nucleotide 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.

[0133] A particular aspect of the recombinant polyadenylation signal sequences according to the present invention is that they function in eukaryotic cells. For example, the recombinant polyadenylation signal sequences provided herein are recognized by an RNA cleavage complex. In some aspects, the RNA cleavage complex is a eukaryotic RNA cleavage complex. In some embodiments, the recombinant polyadenylation signal sequence comprises TG and T-rich domains. In some embodiments, the recombinant polyadenylation signal sequence comprises the nucleotide sequence AATAAA (SEQ ID NO:18). In some embodiments, the recombinant polyadenylation signal sequence comprises a G / T-rich sequence approximately 30 nucleotides downstream of the nucleotide sequence AATAAA (SEQ ID NO:18). In some embodiments, the recombinant polyadenylation signal sequence, when present in an RNA molecule, is capable of binding a cleavage polyadenylation-specific factor (CPSF). In some embodiments, the recombinant polyadenylation signal sequence, when present in an RNA molecule, is capable of binding a cleavage stimulating factor (CstF). In some embodiments, at least one polyadenylation signal sequence results in the termination and polyadenylation of an mRNA transcript operatively linked to at least one polyadenylation signal sequence.

[0134] In some embodiments, a recombinant transcription unit comprising a nucleotide sequence encoding a polypeptide is provided, wherein the nucleotide sequence is operatively linked to a recombinant polyadenylation signal sequence, wherein the recombinant polyadenylation signal sequence comprises or is composed of a nucleotide sequence selected from or consisting of 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.

[0135] In a preferred embodiment, the recombinant polyadenylation signal sequence comprises or consists of a nucleotide sequence selected from or composed of the group consisting of SEQ ID NO:6, SEQ ID NO:9 and SEQ ID NO:12.

[0136] In some embodiments, a recombinant nucleic acid comprising at least one polyadenylation signal sequence provided herein is provided. A common obstacle known in the art when multiple regulatory elements (such as polyadenylation signal sequences) are required in a recombinant nucleic acid (such as one or more plasmids) is that closely spaced identical sequences can lead to recombination events. Therefore, it is advantageous if such recombination events can be reduced or omitted. One aspect of the invention is a plurality of novel short recombinant polyadenylation signal sequences having (shared) low sequence homology (low sequence identity) with each other. Furthermore, the recombinant polyadenylation signal sequence influences the strong expression of a nucleotide sequence encoding a target polypeptide operably linked to the recombinant polyadenylation signal sequence.

[0137] In some embodiments, the recombinant nucleic acid comprises more than one recombinant polyadenylation signal sequence provided herein. In some embodiments, the recombinant nucleic acid comprises two or more recombinant polyadenylation signal sequences provided herein. In some embodiments, the recombinant nucleic acid comprises three recombinant polyadenylation signal sequences provided herein. In a preferred embodiment, the recombinant nucleic acid comprises the polyadenylation signal sequences of SEQ ID NO:6, SEQ ID NO:9, and SEQ ID NO:12. In a particular such embodiment, the recombinant nucleic acid comprises three separate polyadenylation signal sequences, wherein the first polyadenylation signal sequence consists of the nucleotide sequence of SEQ ID NO:6, the second polyadenylation signal sequence consists of the nucleotide sequence of SEQ ID NO:9, and the third polyadenylation signal sequence consists of the nucleotide sequence of SEQ ID NO:12.

[0138] In one embodiment, a recombinant nucleic acid is provided, comprising:

[0139] (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

[0140] (b) A second recombinant transcription unit comprising a second nucleotide sequence encoding a second polypeptide operably linked to a second recombinant polyadenylation signal sequence.

[0141] The first recombinant polyadenylation signal sequence and the second recombinant polyadenylation signal sequence have less than 70%, 65%, 60%, 55%, 50%, 45%, or 40% sequence identity.

[0142] In one embodiment, the recombinant nucleic acid further comprises:

[0143] (c) A third recombinant transcription unit comprising a third nucleotide sequence encoding a third polypeptide that is operatively linked to a third recombinant polyadenylation signal sequence.

[0144] The first and second recombinant polyadenylation signal sequences individually share less than 70%, 65%, 60%, 55%, 50%, 45%, or 40% sequence identity with the third recombinant polyadenylation signal sequence.

[0145] In one embodiment, the recombinant nucleic acid further comprises:

[0146] (c) A third recombinant transcription unit comprising a third nucleotide sequence encoding a third polypeptide that is operatively linked to a third recombinant polyadenylation signal sequence.

[0147] The first recombinant polyadenylation signal sequence and the third recombinant polyadenylation signal sequence have less than 70%, 65%, 60%, 55%, 50%, 45% or 40% sequence identity.

[0148] In one embodiment, the recombinant nucleic acid further comprises:

[0149] (c) A third recombinant transcription unit comprising a third nucleotide sequence encoding a third polypeptide that is operatively linked to a third recombinant polyadenylation signal sequence.

[0150] The second recombinant polyadenylation signal sequence and the third polyadenylation signal sequence have less than 70%, 65%, 60%, 55%, 50%, 45% or 40% sequence identity.

[0151] In some embodiments, 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, 99, 98, 97, or 96 nucleotides. In some embodiments, 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.

[0152] The term "percentage (%) sequence identity" is defined as the percentage of nucleotides in the target sequence that are identical to those in the candidate sequence after sequence alignment, with gaps introduced where necessary to achieve maximum percentage sequence identity. Alignment can be achieved in various ways 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 aligning sequences, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0153] In some respects, the percentage (%) sequence identity of the first recombinant polyadenylation signal sequence and the second recombinant polyadenylation signal sequence is determined by the following:

[0154] (a) The sequences of the first and second recombinant polyadenylation signal sequences were compared using Align-2 software and standard settings.

[0155] (b) Determine the percentage of nucleotides in the first recombinant polyadenylation signal sequence that are identical to those in the second recombinant polyadenylation signal sequence to obtain percentage (%) sequence identity.

[0156] In some respects, the percentage (%) sequence identity of the first and second recombinant polyadenylation signal sequences with the third recombinant polyadenylation signal sequence is determined by the following methods:

[0157] (a) The sequences of the first and third recombinant polyadenylation signal sequences were compared using Align-2 software and standard settings.

[0158] (b) Determine the percentage of nucleotides in the first recombinant polyadenylation signal sequence that are identical to those in the third recombinant polyadenylation signal sequence to obtain the percentage (%) sequence identity between the first and third recombinant polyadenylation signal sequences.

[0159] (c) The sequences of the second and third recombinant polyadenylation signal sequences were compared using Align-2 software and standard settings.

[0160] (d) Determine the percentage of the nucleotides in the second recombinant polyadenylation signal sequence that are identical to those in the third recombinant polyadenylation signal sequence to obtain the percentage (%) sequence identity of the second and third recombinant polyadenylation signal sequences.

[0161] Recombination events between nucleic acids (e.g., plasmids) can occur through a variety of mechanisms, including homologous recombination and site-specific 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 plasmid / chromosome may have different genetic contents and may confer different functions to the cell. In the context of this invention, such recombination events are undesirable, and the inventors seek to provide new and improved sequences to reduce or suppress recombination events. Therefore, in some aspects, recombination events between nucleic acids containing a first recombination polyadenylation signal sequence and nucleic acids containing a second recombination polyadenylation signal sequence (and, if present, a third recombination polyadenylation signal sequence) are reduced or prevented.

[0162] The inventors have developed 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 aspects, the first polyadenylation signal sequence, the second polyadenylation signal sequence, and, if present, a third polyadenylation signal sequence, cannot participate in DNA strand exchange to form a recombination intermediate. “DNA strand exchange” is a key step in the process of mutual recombination. Two DNA molecules break at corresponding sites and exchange fragments of their strands with each other, then rejoin to form two new hybrid DNA molecules. DNA strand exchange involves the formation of heteroduplex structures, in which the single-stranded ends of the broken polynucleotide molecules invade each other's double helixes and form base-pairing regions (Holliday linkers) between the two molecules. This “recombination intermediate” allows DNA strands to cross over each other, facilitating the exchange of DNA fragments between the two molecules. Subsequently, the Holliday linker can be broken down by strand cleavage, leading to the formation of hybrid DNA molecules.

[0163] The formation of Holliday linkers during homologous recombination requires a significant degree of sequence homology between the two DNA molecules involved in the exchange. Specifically, the homologous sequences must be long enough and have a sufficiently high degree of similarity to form a stable heteroduplex DNA structure. Without theoretical constraints, the minimum length and degree of homology required to form Holliday linkers can vary depending on the DNA molecules involved and the specific enzymes and cofactors involved. Generally, it is believed that at least 100-200 base pairs of consecutive homologous DNA sequences are needed to form stable Holliday linkers.

[0164] In some aspects, recombination events between nucleic acids containing a first polyadenylation signal sequence and nucleic acids containing a second polyadenylation signal sequence are reduced or prevented. In some aspects, recombination events between nucleic acids containing a first polyadenylation signal sequence and nucleic acids containing a third polyadenylation signal sequence are reduced or prevented. In some embodiments, recombination events between nucleic acids containing a second polyadenylation signal sequence and nucleic acids containing a third polyadenylation signal sequence are reduced or prevented.

[0165] Recombination events can be detected using methods known in the art. For example, recombination events can be detected by Sanger sequencing of the relevant PCR amplicons, followed by sequence alignment (e.g., using CLUSTALW) and identification of the recombination event (e.g., using a recombination detection procedure). In some aspects, recombination events are detected by Sanger sequencing of PCR amplicons containing the recombinant polyadenylation signal sequence provided herein, followed by alignment of the PCR amplicons with CLUSTALW using standard settings, and identification of the recombination event using recombination detection procedure 5 using standard settings. In a preferred aspect, no recombination event is detected.

[0166] The recombinant polyadenylation signal sequence of the present invention can be used in various applications. Polyadenylation signal sequences are essential for efficient protein expression. Therefore, in some aspects, the recombinant transcription unit according to the present invention is capable of driving the expression of a nucleotide sequence encoding a target polypeptide. In some aspects, the nucleotide sequence encoding the target polypeptide is operatively linked to the recombinant transcription unit. In some aspects, the nucleotide sequence encoding the target polypeptide is operatively linked to the recombinant polyadenylation signal sequence provided herein.

[0167] In some aspects, the first recombinant transcription unit, the second recombinant transcription unit, and, if present, the third recombinant transcription unit are active in eukaryotic cells. In some aspects, the first polypeptide, the second polypeptide, and, if present, the third polypeptide are expressed by eukaryotic cells. In some aspects, eukaryotic cells are incubated under conditions suitable for the expression of the first polypeptide, the second polypeptide, and, if present, the third polypeptide. In some aspects, eukaryotic cells are cultured under conditions suitable for the expression of the first polypeptide, the second polypeptide, and, if present, the third polypeptide. In some aspects, a method for producing one (or more) polypeptides is provided, the method comprising the step of culturing a host cell containing at least one recombinant transcription unit as described herein under conditions suitable for the expression of one (or more) polypeptides.

[0168] Protein expression can be measured by assays readily available in the art, such as those described in the examples provided below.

[0169] The terms plasmid, construct, and vector are used throughout this specification. As used herein, the term "plasmid" refers to a circular supercoiled DNA molecule that assembles 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 uses them as a vector to express genes in a vertebrate host. Furthermore, as used herein, "plasmid" is capable of replicating in bacterial strains. As used herein, the term "construct" refers to a specific vector or plasmid having a specific gene arrangement and regulatory elements. Nucleic acid sequences can be "exogenous," meaning they are foreign to the cells to which the vector is introduced; "heterogeneous," meaning they originate from a different genetic source; or "homogeneous," meaning the sequence is structurally related to a sequence in the cell but is not typically found in the host cell's nucleic acid. Methods for constructing vectors or modifying plasmids of the present invention using standard recombination techniques are well known in the art, for example, as described in Sambrook et al., Molecular Cloning. A Laboratory Manual, Cold SpringHarbor 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.

[0170] The term "vector" is used to refer to a vector nucleic acid molecule into which a designated nucleic acid molecule encoding one or more antigens can be inserted to introduce it into cells capable of expression. Vectors include plasmids, glial bodies, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YAC). The term "expression vector" refers to a vector containing a nucleic acid sequence encoding a gene product that can be transcribed at least partially. 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), short interfering RNA (siRNA), antisense molecules, or ribozymes. Expression vectors may contain a variety of "control sequences," which refer to nucleic acid sequences operatively linked in a particular host organism that are essential for transcription and possible translation of the coding sequence. In addition to control sequences that control transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that perform other functions, as described below.

[0171] It should be understood that, in order to prevent recombination events within nucleic acids containing multiple transcription units, other elements of the transcription unit, besides the recombination polyadenylation signal sequence, can also form recombination intermediates that may lead to recombination events. Therefore, it is preferred that different recombination transcription units do not contain elements with high sequence homology. It should also be understood that, generally, nucleotide sequences encoding a target polypeptide do not share high sequence homology. However, in cases where closely related genes encoding a target polypeptide are included in different transcription units contained in nucleic acids as provided herein, it is preferred that the nucleotide sequences encoding different polypeptides have sequence identity of less than 70%, 65%, 60%, 55%, 50%, 45%, or 40%. Other elements of the transcription unit that can form recombination intermediates are promoters contained within the transcription unit. In some aspects, the nucleic acid includes a first promoter and a second promoter. In some aspects, the first promoter and the second promoter are not the same promoter. In some aspects, the nucleic acid further includes a third promoter. In some aspects, the third promoter is not the same promoter as the first promoter and / or the second promoter.

[0172] In one embodiment, a recombinant nucleic acid as described above is provided, wherein

[0173] (a) The first recombinant transcription unit further comprises a first promoter operatively linked to a nucleotide sequence encoding a first polypeptide, and

[0174] (b) The second recombinant transcription unit further comprises a second promoter operatively linked to a nucleotide sequence encoding a second polypeptide.

[0175] The first promoter and the second promoter have sequence identity of less than 70%, 65%, 60%, 55%, 50%, 45%, or 40%.

[0176] In one embodiment, the recombinant nucleic acid further comprises:

[0177] (c) If the first recombinant transcription unit further comprises a first promoter operatively linked to a nucleotide sequence encoding a first polypeptide,

[0178] The first and second promoters have sequence identity with the third promoter of less than 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 65%, or 60%.

[0179] In one embodiment, a recombinant nucleic acid is provided, comprising:

[0180] (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 a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%; 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence of SEQ ID NO:6, and

[0181] (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 a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%; 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence of SEQ ID NO:9, optionally

[0182] Eukaryotic cells transformed with a recombinant nucleic acid containing a first recombinant transcription unit are able to express the polypeptide at the same or higher expression level as the polypeptide in eukaryotic cells transformed with a reference nucleic acid containing a recombinant polyadenylation signal sequence consisting of SEQ ID NO:2, wherein the nucleotide sequence of the reference nucleic acid is identical to that of the recombinant nucleic acid, without regard to sequence identity.

[0183] Eukaryotic cells transformed with a recombinant nucleic acid containing a second recombinant transcription unit are able to express the polypeptide at the same or higher expression level as the polypeptide in eukaryotic cells transformed with a reference nucleic acid containing a recombinant polyadenylation signal sequence consisting of SEQ ID NO:2, wherein the nucleotide sequence of the reference nucleic acid is identical to that of the recombinant nucleic acid without regard to sequence identity.

[0184] In one embodiment, a recombinant nucleic acid is provided, comprising:

[0185] (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 a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%; 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence of SEQ ID NO:6, and

[0186] (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 a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%; 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence of SEQ ID NO:12, optionally

[0187] Eukaryotic cells transformed with a recombinant nucleic acid containing a first recombinant transcription unit are able to express the polypeptide at the same or higher expression level as the polypeptide in eukaryotic cells transformed with a reference nucleic acid containing a recombinant polyadenylation signal sequence consisting of SEQ ID NO:2, wherein the nucleotide sequence of the reference nucleic acid is identical to that of the recombinant nucleic acid, without regard to sequence identity.

[0188] Eukaryotic cells transformed with a recombinant nucleic acid containing a second recombinant transcription unit are able to express the polypeptide at the same or higher expression level as the polypeptide in eukaryotic cells transformed with a reference nucleic acid containing a recombinant polyadenylation signal sequence consisting of SEQ ID NO:2, wherein the nucleotide sequence of the reference nucleic acid is identical to that of the recombinant nucleic acid without regard to sequence identity.

[0189] In one embodiment, a recombinant nucleic acid is provided, comprising:

[0190] (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 a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%; 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence of SEQ ID NO:9, and

[0191] (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 a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%; 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence of SEQ ID NO:12, optionally

[0192] Eukaryotic cells transformed with a recombinant nucleic acid containing a first recombinant transcription unit are able to express the polypeptide at the same or higher expression level as the polypeptide in eukaryotic cells transformed with a reference nucleic acid containing a recombinant polyadenylation signal sequence consisting of SEQ ID NO:2, wherein the nucleotide sequence of the reference nucleic acid is identical to that of the recombinant nucleic acid, without regard to sequence identity.

[0193] Eukaryotic cells transformed with a recombinant nucleic acid containing a second recombinant transcription unit are able to express the polypeptide at the same or higher expression level as the polypeptide in eukaryotic cells transformed with a reference nucleic acid containing a recombinant polyadenylation signal sequence consisting of SEQ ID NO:2, wherein the nucleotide sequence of the reference nucleic acid is identical to that of the recombinant nucleic acid without regard to sequence identity.

[0194] In one embodiment, a recombinant nucleic acid is provided, comprising:

[0195] (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 is composed of the nucleotide sequence of SEQ ID NO:6, and

[0196] (b) A second recombinant transcription unit comprising a second nucleotide sequence encoding a second polypeptide operatively linked to a second recombinant polyadenylation signal sequence, wherein the first recombinant polyadenylation signal sequence comprises or is composed of the nucleotide sequence of SEQ ID NO:9.

[0197] In one embodiment, a recombinant nucleic acid is provided, comprising:

[0198] (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 is composed of the nucleotide sequence of SEQ ID NO:6, and

[0199] (b) A second recombinant transcription unit comprising a second nucleotide sequence encoding a second polypeptide operatively linked to a second recombinant polyadenylation signal sequence, wherein the first recombinant polyadenylation signal sequence comprises or is composed of the nucleotide sequence of SEQ ID NO:12.

[0200] In one embodiment, a recombinant nucleic acid is provided, comprising:

[0201] (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 is composed of the nucleotide sequence of SEQ ID NO:9, and

[0202] (b) A second recombinant transcription unit comprising a second nucleotide sequence encoding a second polypeptide operatively linked to a second recombinant polyadenylation signal sequence, wherein the first recombinant polyadenylation signal sequence comprises or is composed of the nucleotide sequence of SEQ ID NO:12.

[0203] In one embodiment, a recombinant nucleic acid is provided, comprising:

[0204] (a) A first recombinant transcription unit comprising a first nucleotide sequence encoding a first polypeptide operatively linked to a first recombinant polyadenylation signal sequence, wherein the first recombinant polyadenylation signal sequence comprises or is composed of a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO:6.

[0205] (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 a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%; 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence of SEQ ID NO:9, and

[0206] (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 a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%; 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence of SEQ ID NO:12, optionally

[0207] Eukaryotic cells transformed with a recombinant nucleic acid containing a first recombinant transcription unit are able to express the polypeptide at the same or higher expression level as the polypeptide in eukaryotic cells transformed with a reference nucleic acid containing a recombinant polyadenylation signal sequence consisting of SEQ ID NO:2, wherein the nucleotide sequence of the reference nucleic acid is identical to that of the recombinant nucleic acid, without considering the recombinant polyadenylation signal sequence for sequence identity.

[0208] Eukaryotic cells transformed with a recombinant nucleic acid containing a second recombinant transcription unit are able to express the polypeptide at the same or higher expression level as the polypeptide in eukaryotic cells transformed with a reference nucleic acid containing a recombinant polyadenylation signal sequence consisting of SEQ ID NO:2, wherein the nucleotide sequence of the reference nucleic acid is identical to that of the recombinant nucleic acid, without considering the recombinant polyadenylation signal sequence for sequence identity.

[0209] Eukaryotic cells transformed with a recombinant nucleic acid containing a third recombinant transcription unit are able to express the polypeptide at the same or higher expression level as the polypeptide in eukaryotic cells transformed with a reference nucleic acid containing a recombinant polyadenylation signal sequence consisting of SEQ ID NO:2, wherein the nucleotide sequence of the reference nucleic acid is identical to that of the recombinant nucleic acid without regard to sequence identity.

[0210] In one embodiment, a recombinant nucleic acid is provided, comprising:

[0211] (a) A first recombinant transcription unit comprising a first nucleotide sequence encoding a first polypeptide operatively linked to a first recombinant polyadenylation signal sequence, wherein the first recombinant polyadenylation signal sequence comprises or is composed of the nucleotide sequence of SEQ ID NO:6.

[0212] (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 is composed of the nucleotide sequence of SEQ ID NO:9, and

[0213] (c) A third recombinant transcription unit comprising a third nucleotide sequence encoding a third polypeptide operatively linked to a third recombinant polyadenylation signal sequence, wherein the third recombinant polyadenylation signal sequence comprises or is composed of the nucleotide sequence of SEQ ID NO:12.

[0214] In some aspects, the first promoter, the second promoter, and, if present, the third promoter are active in eukaryotic cells. In some aspects, the first promoter, the second promoter, and, if present, the third promoter are capable of driving the expression of the target peptide in eukaryotic cells. The expression of the target peptide can be measured by assays readily available in the art, such as those described in the examples provided below. In some aspects, the first promoter drives the expression of the first peptide. In some aspects, the second promoter drives the expression of the second peptide. In some aspects, the third promoter drives the expression of the third peptide. In some aspects, the first promoter is capable of driving the expression of the first peptide, and the second promoter is capable of driving the expression of the second peptide, and, if present, the third promoter is capable of driving the expression of the third peptide.

[0215] The term "promoter" refers to a polynucleotide sequence that controls the transcription of a gene / structural gene or nucleic acid sequence that it is operatively linked to. Promoters include signals for RNA polymerase binding and transcription initiation. The promoter used will function in the cell intended to express the selected structural gene. A large number of promoters, including constitutive, inducible, and repressive promoters from a wide variety of sources, are well known in the art (and identified in databases such as GenBank) and are available as cloned polynucleotides or within cloned polynucleotides (e.g., from repositories such as ATCC and other commercial or personal sources).

[0216] Typically, promoters are located in the 5' non-coding or untranslated region of a gene, near the transcription start site of the structural gene. The sequence elements intrinsically linked to the promoter in transcription initiation are usually characterized by shared nucleotide sequences. These elements include RNA polymerase binding sites, TATA sequences, CAAT sequences, differentiation-specific elements (DSEs), circular 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 inducible, the transcription rate increases in response to an inducer, such as the CMV promoter, followed by two tet-operon sites, metallothionein, and the heat shock promoter. If the promoter is constitutively active, the transcription rate is not regulated by an inducer. Exemplary eukaryotic promoters identified as strong promoters of expression are the SV40 early promoter, the adenovirus major late promoter, the mouse metallothionein-I promoter, the Rous sarcoma virus long terminal repeat sequence, the Chinese hamster elongation factor 1α (CHEF-1), human EF-1α, ubiquitin, and the human cytomegalovirus major early-mid promoter (hCMV MIE).

[0217] In some respects, the first promoter, the second promoter, and, if present, the third promoter are selected from the SV40 early promoter, the adenovirus major late promoter, the mouse metallothionein-I promoter, the Rous sarcoma virus long terminal repeat sequence, the Chinese hamster elongation factor 1α (CHEF-1), human EF-1α, ubiquitin, and the human cytomegalovirus major early-mid promoter (hCMV MIE).

[0218] The nucleic acids according to the present invention may be contained in a vector or multiple vectors.

[0219] Therefore, this disclosure also provides one or more vectors comprising the nucleic acid or multiple nucleic acids according to the invention. The vector facilitates the delivery of nucleic acid encoding one or more recombinant transcription units to cells. The vector may be an expression vector containing elements required for expressing recombinant polypeptides according to the invention. The vector may contain elements facilitating the integration of the nucleic acid into the genomic DNA of the cell in which the vector is introduced.

[0220] The nucleic acids and vectors disclosed herein can be provided in purified or isolated form, i.e., derived from other nucleic acids or naturally occurring biological materials.

[0221] The vector can be a vector for expressing nucleic acids in cells (i.e., an expression vector). Such vectors may include a promoter sequence operatively linked to a nucleotide sequence encoding a recombinant polypeptide according to the present disclosure. The vector may also include a stop codon (i.e., located at the 3' of the nucleotide sequence encoding the recombinant polypeptide in the nucleotide sequence of the vector) and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon known in the art may be used to express peptides or polypeptides from vectors according to the present disclosure.

[0222] Vectors intended to be associated with this disclosure include DNA vectors, RNA vectors, plasmids (e.g., conjugating plasmids (e.g., F plasmids), non-conjugating plasmids, R plasmids, col plasmids, episomes), viral vectors (e.g., retroviral vectors, such as gamma retroviral vectors (e.g., vectors derived from murine leukemia virus (MLV), such as 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), as described, for example, in Maus et al., Annu Rev Immunol (2014) 32:189-225 and Morgan and Boyerinas, Biomedicines (2016) 4:9, the entire contents of which are incorporated herein by reference. In some embodiments, the vectors according to this disclosure are lentiviral vectors.

[0223] In some aspects, the vector can be a eukaryotic vector, i.e., a vector containing elements necessary for protein expression from a eukaryotic cell. In some embodiments, the vector can be a mammalian vector, which, for example, contains a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.

[0224] In some aspects, the first vector, the second vector, and / or, if present, the third vector, contains a bacterial origin for replication. In some aspects, the first vector contains a bacterial origin for replication. In some aspects, the second vector contains a bacterial origin for replication. In some aspects, the third vector contains a bacterial origin for replication. Replication of a vector (such as a plasmid) in bacteria requires a bacterial origin for replication. The bacterial origin for replication is known in the art. In some aspects, the replicating bacterial primordium is the pUC origin of replication.

[0225] In some aspects, the recombinant nucleic acid provided herein comprises a first vector as described above, which contains a first recombinant transcription unit as described above; a second vector as described above, which contains a second recombinant transcription unit as described above; and, if present, a third recombinant transcription unit, which contains a third recombinant transcription unit as described above. The recombinant nucleic acid may be provided in one or more vials. For example, the first vector, the second vector, and, if present, the third vector may be provided together in one vial. Alternatively, the first vector, the second vector, and, if present, the third vector may be provided in separate vials. In some aspects, the first vector, the second vector, and, if present, the third vector are provided in separate vials, but the vials together constitute the recombinant nucleic acid provided herein. In some aspects, the first vector, the second vector, and, if present, the third vector are provided in the same vial.

[0226] In some respects, recombinant nucleic acids contain at least one selectable biomarker. In other respects, vectors, as described above, contain selectable biomarkers. The term "selectable biomarker" refers to a nucleic acid that allows cells carrying it to be specifically selected for or against in the presence of a corresponding selector. Typically, a selectable biomarker will confer resistance to a drug or compensate for metabolic or catabolistic defects in the introduced cells. Selectable biomarkers can be positive, negative, or bifunctional. A useful positive selectable biomarker is an antibiotic resistance gene, which allows selection of cells transformed with a corresponding selector (e.g., an antibiotic) in the presence of a selector. Untransformed cells cannot grow or survive under selective conditions, i.e., in the presence of the selector. A negative selectable biomarker allows for the selective elimination of cells carrying the biomarker. Selectable markers for use with eukaryotic cells include, for example, structural genes encoding aminoglycoside phosphotransferases (APH), such as hygromycin (hyg), neomycin (neo), and G418 selectable markers, dihydrofolate reductase (DHFR), thymidine kinase (tk), glutamine synthase (GS), asparagine synthase, tryptophan synthase (selector indole), histidine dehydrogenase (selector histidine D), and nucleic acids conferring resistance to purines, bleomycin, humulin, chloramphenicol, bleomycin, and mycophenolic acid.

[0227] In some respects, the selectable biomarkers are selected from hygromycin selectable biomarkers, neomycin selectable biomarkers, G418 selectable biomarkers, dihydrofolate reductase (DHFR), thymidine kinase, glutamine synthase, asparagine synthase, tryptophan synthase, histidine dehydrogenase, and nucleic acids that confer resistance to puromycin, bleomycin, fulvicin, chloramphenicol, bleomycin, and mycophenolic acid.

[0228] In some respects, recombinant nucleic acids contain at least one bacterial origin of replication. In other respects, vectors, as described above, contain the bacterial origin of replication. For vectors / plasmids to replicate independently within bacterial cells, they must have a segment of DNA that can serve as an origin of replication. The origin of replication (also called the replication origin) is a specific sequence that initiates replication. Exemplary origins of replication may be derived from the pUC plasmid cloning vector created by Joachim Messing and colleagues (Yanisch-Perron, C.; Vieira, J.; Messing, J. (1985).. Gene. 33 (1): 103–119), and in some respects, the first vector, the second vector, and / or, if present, the third vector, contain the bacterial origin of replication, particularly the pUC19 origin of replication.

[0229] It should be understood that the nucleic acids of the present invention can be used for the recombinant production of proteins. As described above, in the context of recombinant polypeptide expression, the use of novel recombinant polyadenylation signal sequences is advantageous to mitigate the risk of recombination events occurring between highly homologous or identical sequences.

[0230] Therefore, it is further provided that cells (e.g., host cells) contain recombinant nucleic acids according to the present invention.

[0231] In some respects, the cell is the host cell. The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including progeny of such cells. Host cells include “transformations” and “transformed cells,” which include primary transformed cells and progeny derived from those primary transformed cells, regardless of passage number. Progeny may not be identical to the nucleic acid contents of the parent cells and may contain mutations. This article includes mutant progeny with the same function or biological activity as those screened or selected in the original transformed cells.

[0232] To produce a recombinant target protein, the nucleic acid encoding the target protein is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using standard procedures, produced via recombinant methods, or obtained through chemical synthesis.

[0233] Suitable host cells for cloning or expressing the target protein include prokaryotic or eukaryotic cells as described herein. For example, (recombinant) peptides can be generated in bacteria, particularly when glycosylation and Fc effector function are not required. For information on the expression of antibody fragments and peptides in bacteria, see, for example, US 5,648,237, US 5,789,199, and US 5,840,523. (See also Charlton, KA, in: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, describing the expression of antibody fragments in *E. coli*). The target protein can be separated from the bacterial cell paste in a soluble fraction after expression and can be further purified.

[0234] Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable cloning or expression hosts for vectors encoding recombinant peptides, including fungal and yeast strains whose glycosylation pathways have been “humanized,” resulting in the production of peptides with partially or fully human glycosylation patterns. See Gerngross, TU, Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.

[0235] Suitable host cells for expressing (glycosylated) peptides also originate 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 conjunction with insect cells, particularly for transfecting cells of the meadow armyworm (Spodoptera frugiperda).

[0236] Plant cell cultures can also be used as hosts. See, for example, US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978 and US 6,417,429 (which describe PLATNIBODIES™ technology for producing antibodies in transgenic plants).

[0237] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be useful. Other examples of useful mammalian host cell lines include monkey kidney CV1 (COS-7) transformed with SV40; human embryonic kidney (HEK) cell lines (such as 293 or 293T cells described, for example, in Graham, FL et al., J. Gen Virol. 36 (1977) 59-74); hamster kidney cells (BHK); mouse Sertoli cells (such as TM4 cells described, for example, in 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 (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (MMT 060562); and TRI cells (such as those described, for example, in Mather, JP et al., Annals). (As described in NY Acad. Sci. 383 (1982) 44-68); MRC 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, NSO, and Sp2 / 0. For a review of certain 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.

[0238] In some respects, the (host) cell is a eukaryotic cell. In some respects, the (host) cell is a eukaryotic host cell. In some respects, the (host) cell is a mammalian host cell. In some respects, the (host) cell is selected from the group consisting of CHO, BHK, HEK, and Sp2 / O. In some respects, the (host) cell is CHO K1.

[0239] Therefore, in one embodiment, a method for generating a polypeptide is provided, comprising the following steps:

[0240] (a) Provide a host cell containing the recombinant nucleic acid as described above,

[0241] (b) Incubate host cells under conditions suitable for peptide expression.

[0242] (c) Recover the target peptide from the cell culture.

[0243] In one embodiment, a method for generating a polypeptide is provided, comprising the following steps:

[0244] (a) A cell comprising the recombinant nucleic acid as described above, wherein the recombinant nucleic acid comprises at least one polyadenylation signal sequence, wherein the at least one polyadenylation signal sequence is operatively linked to a nucleotide sequence encoding a polypeptide.

[0245] (b) Incubate cells under conditions suitable for peptide expression.

[0246] (c) Recover the target peptide from the cell culture.

[0247] In one embodiment, a method for generating a target peptide is provided, the method comprising the following steps:

[0248] (a) Providing a host cell comprising the recombinant nucleic acid as described above, wherein the target polypeptide is a first polypeptide, and wherein a second polypeptide and, if present, a third polypeptide are necessary for or improve the production of the target polypeptide.

[0249] (b) Incubate host cells under conditions suitable for the expression of the first polypeptide, the second polypeptide, and, if present, a third polypeptide.

[0250] (c) Recovery of the target peptide from cell cultures, and optionally

[0251] (d) Formulate the recovered target peptide for therapeutic use.

[0252] Further, this invention provides a method for producing viral vectors using the recombinant polyadenylation signal sequence of the present invention. Producing viral vectors using multiple separate plasmids is a widely used and efficient technique that can generate high-quality viral particles for research and clinical applications. However, due to the use of multiple plasmids, it is necessary to mitigate the possibility of recombination events between highly homologous or identical sequence extensions on different plasmids. The polyadenylation signal sequence according to the present invention is advantageous in this context. Furthermore, the size of a viral vector genome is typically limited. Therefore, it is advantageous to integrate short 5' and 3' regulatory sequences to maximize the sequence length available for (therapeutic) transgenesis.

[0253] In some respects, recombinant viral vectors containing the recombinant polyadenylation signal sequence as described above are provided.

[0254] In some aspects, a recombinant viral vector is provided that contains a capsid and a vector genome package. In some embodiments, viral vectors that can be used in this invention include, but are not limited to, retroviruses, adenoviruses, helper-dependent adenoviruses, hybrid adenoviruses, herpes simplex virus, lentiviruses, poxviruses, Epstein-Barr virus, vaccinia virus, and human cytomegalovirus vectors, including recombinant versions thereof. In preferred embodiments, the recombinant viral vector comprises a lentiviral vector, an adenovirus vector, or an adeno-associated virus (AAV) vector. In some aspects, the recombinant viral vector is a recombinant adeno-associated virus (rAAV) containing an adeno-associated virus (AAV) capsid and a vector genome packaged therein.

[0255] In one embodiment, a recombinant viral vector comprising a vector genome is provided, wherein the vector genome is comprised in a 5' to 3' sequence:

[0256] (i) 5' ITR sequence,

[0257] (ii) Startup subsequence

[0258] (iii) The sequence encoding the polypeptide.

[0259] (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

[0260] (v)3' ITR sequence.

[0261] In a preferred embodiment, 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.

[0262] The term “recombinant” as a modifier for viral vectors (such as recombinant AAV (rAAV) vectors) refers to a composition that has been manipulated (i.e., engineered) in a manner not normally found in nature. A specific example of a recombinant AAV vector is the insertion of nucleic acids (heteronucleotides) not normally present in the wild-type AAV genome into the viral genome. One example is the cloning of nucleic acids (e.g., genes) encoding therapeutic proteins or polynucleotide sequences into a vector, with or without the 5', 3', and / or intron regions that genes typically associate with within the AAV genome. Although the term “recombinant” is not always used to refer to AAV vectors, recombinant forms are explicitly included despite any such omissions.

[0263] For example, an "rAAV vector" is derived from the wild-type genome of AAV. This is achieved by removing all or part of the wild-type AAV genome using molecular methods and replacing it with non-natural (heterologous) nucleic acids, such as those encoding therapeutic proteins or polynucleotide sequences. Typically, for an rAAV vector, one or both inverted terminal repeats (ITRs) of the AAV genome are preserved. rAAV differs from the AAV genome because all or part of the AAV genome has been replaced by non-natural sequences of AAV genome nucleic acids, such as heterologous nucleic acids encoding therapeutic proteins or polynucleotide sequences. Therefore, the combination of non-natural (heterologous) sequences defines AAV as a "recombinant" AAV vector, which may be called an "rAAV vector."

[0264] In some respects, eukaryotic cells containing the vector genome described above are capable of expressing polypeptides. In other respects, recombinant polyadenylation signaling sequences lead to the termination (transcription) and polyadenylation of mRNA transcripts in eukaryotic cells.

[0265] Recombinant AAV vector sequences (referred to herein as "particles") can be packaged for subsequent cell infection (transduction) in vitro, in vitro, or in vivo. When the recombinant vector sequence is encapsulated or packaged into an AAV particle, the particle may also be referred to as "rAAV," "rAAV particle," and / or "rAAV viral particle." Such rAAV, rAAV particles, and rAAV viral particles include proteins that encapsulate or package the vector genome. In the case of AAV, specific instances include capsid proteins.

[0266] The "vector genome," abbreviated as "vg," refers to the portion of the recombinant plasmid sequence that is ultimately packaged or encapsulated 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," which is important for plasmid cloning and amplification (the processes required for propagation and recombinant AAV vector production), but it is not itself packaged or encapsulated into rAAV particles. Therefore, the "vector genome" refers to the nucleic acids packaged or encapsulated by rAAV.

[0267] As used herein, the term "serotype," when referring to AAV vectors, refers to a capsid that is serologically distinct from other AAV serotypes. Serological distinctiveness is determined based on the lack of cross-reactivity between antibodies against one AAV and antibodies against another. Differences in cross-reactivity are typically due to differences in capsid protein sequence / antigenic determinants (e.g., due to differences in the VP1, VP2, and / or VP3 sequences of AAV serotypes). Due to homology in capsid protein sequences, antibodies against one AAV can cross-react with one or more other AAV serotypes.

[0268] Under the traditional definition, a serotype refers to a serum that has been tested against a target virus, which is specific for neutralizing activity against all existing and characterized serotypes, and for which no antibodies neutralizing the target virus have been found. As more naturally occurring viral isolates are discovered and / or capsid mutants are generated, serological differences with any of the currently existing serotypes may or may not exist. Therefore, in cases where a new virus (e.g., AAV) does not exhibit serological differences, that new virus (e.g., AAV) will be a subgroup or variant of the corresponding serotype. In many cases, serological tests for neutralizing activity have not been performed on mutant viruses with capsid sequence modifications to determine whether they belong to another serotype according to the traditional definition of serotype. Therefore, for convenience and to avoid duplication, the term "serotype" broadly refers to both serologically distinct viruses (e.g., AAV) and serologically indistinguishable viruses (e.g., AAV), which may fall within a subgroup or variant of a given serotype.

[0269] rAAV viral vectors include any viral strain or serotype. For example, but not limited to, the rAAV vector genome or particle (capsid, such as VP1, VP2, and / or VP3) can be based on any AAV serotype, such as AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -rh74, -rhlO, AAV3B, or AAV-2i8. Such vectors can be based on the same strain or serotype (or subgroup or variant) or different from each other. For example, but not limited to, an rAAV plasmid or vector genome or particle (capsid) based on a serotype genome can be identical to one or more capsid proteins of the packaging vector. Furthermore, the rAAV plasmid or vector genome can be based on an AAV serotype genome of one or more capsid proteins different from the packaging vector genome. In this case, at least one of the three capsid proteins can be a different AAV serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rhlO, AAV3B, AAV-2i8, or a variant thereof. More specifically, the rAAV2 vector genome may contain an AAV2 ITR, but the capsid is derived from a different serotype, such as AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rhlO, AAV3B, AAV-2i8, or a variant thereof. Therefore, rAAV vectors include gene / protein sequences that are identical to those of a specific serotype, as well as “mixed” serotypes, which may also be referred to as “pseudotypes”.

[0270] In some embodiments, the rAAV plasmid or vector genome or particle is based on reptile or invertebrate AAV variants, such as 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).

[0271] In some embodiments, the recombinant plasmid or vector genome or particle is based on a bocavirus variant. For example, human bocavirus variants are described in Guido et al., 2016, World J. Gastroenterol., 22:8684-8697.

[0272] In one embodiment, the recombinant AAV (rAAV) vector comprises VP1, VP2, and / or VP3 capsid proteins having 70% or more sequence identity with the 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, -rhlO, AAV3B, AAV-2i8, VP1, VP2, and / or VP3 capsid proteins. In one embodiment, the recombinant AAV (rAAV) vector comprises VP1, VP2, and / or VP3 capsid proteins having 100% sequence identity with the 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, -rhlO, AAV3B, AAV-2i8, VP1, VP2, and / or VP3 capsid proteins. In some embodiments, the AAV vector comprises or consists of at least 70% or more (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc.) sequences identical to or composed of one or more AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rhlO, or AAV3B, ITR.

[0273] In some embodiments, the recombinant AAV (rAAV) vector includes its AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV3B, RhlO, Rh74, and AAV-2i8 variants (e.g., ITR and capsid variants, such as amino acid insertions, additions, substitutions, and deletions), for example, as described in WO 2013 / 158879 (International Application PCT / US2013 / 037170), WO 2015 / 013313 (International Application PCT / US2014 / 047670), and US 2013 / 0059732 (US Application No. 13 / 594,773).

[0274] rAAVs, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rh10, AAV3B, AAV-2i8 and their variants, hybrid and chimeric sequences, can be constructed using recombinant techniques known to those skilled in the art, including one or more heterologous polynucleotide sequences (transgenics) flanked by one or more functional AAV ITR sequences. Such AAV vectors typically retain at least one functional flanking ITR sequence, which is essential for rescuing, replicating, and packaging the recombinant vector into rAAV vector particles. Therefore, the rAAV vector genome will include the cis sequences (e.g., functional ITR sequences) required for replication and packaging.

[0275] In some respects, the AAV capsid is selected from the group consisting of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10, AAV3B, AAV-2i8 capsids or variant capsids derived therefrom.

[0276] In some respects, recombinant adeno-associated virus (rAAV) comprises a vector genome containing at least one promoter sequence. In some respects, the promoter is selected from the SV40 early promoter, the adenovirus major late promoter, the mouse metallothionein-I promoter, the Rous sarcoma virus long terminal repeat sequence, the Chinese hamster elongation factor 1α (CHEF-1), human EF-1α, ubiquitin, and the human cytomegalovirus major early-mid promoter (hCMV MIE).

[0277] In other respects, methods for generating recombinant adeno-associated virus (rAAV) vectors are provided.

[0278] In one embodiment, a method for generating a recombinant adeno-associated virus (rAAV) vector is provided, the method comprising the following steps:

[0279] (a) Providing a host cell containing the recombinant nucleic acid as described above, wherein a first polynucleotide sequence encodes a therapeutic payload, wherein a second nucleotide sequence encodes the viral vector rep and cap proteins, and wherein a third nucleotide sequence encodes the E4, E2a, and VA proteins.

[0280] (b) Incubate host cells under conditions suitable for generating the recombinant rAAV vector, and

[0281] (c) Recovery of viral vectors from cell cultures, and optionally

[0282] (d) Formulate the recovered target peptide for therapeutic use.

[0283] In the production of rAAV vectors, host cells are used to replicate and package the viral genome into the AAV capsid. For example, human embryonic kidney cells (HEK cells), genetically engineered to produce the necessary proteins 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 with therapeutic genes, as well as the rep and cap genes required for replicating and packaging the viral genome into the capsid. The plasmids provide the necessary genetic material for the production of rAAV particles. The host cells replicate and package the AAV genome into AAV particles, which can then be harvested and purified, for example, for gene therapy. Well-characterized host cells (such as HEK293 cells) can help ensure the consistency and reliability of the rAAV particles. Other cells that can be used in the context of rAAV production are known in the art.

[0284] In some aspects, the host cell is a eukaryotic host cell. In some aspects, the host cell is a mammalian host cell. In some aspects, the host cell is selected from the group consisting of CHO cells, BHK cells, HEK cells, and Sp2 / O cells. In a preferred embodiment, the host cell is an HEK host cell, particularly a HEK293 host cell.

[0285] In some aspects, the polypeptides and rAAV vectors produced according to the invention are further processed, such as, for example, formulated for therapeutic use. Therefore, pharmaceutical compositions comprising polypeptides or rAAV vectors produced according to the invention are also provided herein. In one aspect, the pharmaceutical composition comprises any polypeptide or viral vector provided herein and a pharmaceutically acceptable carrier. In another aspect, the pharmaceutical composition comprises any polypeptide or viral vector provided herein and at least one additional therapeutic agent, such as those described below.

[0286] Pharmaceutical compositions (formulations) can be prepared by combining a peptide or viral vector with a pharmaceutically acceptable carrier or excipient known to those skilled in the art. Exemplary pharmaceutical compositions as described herein are lyophilized, aqueous, frozen, etc.

[0287] Pharmaceutical carriers are generally non-toxic to the treated individual at the dosage and concentration 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 (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl diammonium chloride; benzalkonium chloride; benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol); and low molecular weight (less than about 10). (1 residue) polypeptides; proteins, such as 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., zinc protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG).

[0288] Pharmaceutical compositions intended for internal administration are typically sterile. For example, sterility can be readily achieved through filtration using a sterile filter membrane.

[0289] Any polypeptide or viral vector produced according to the present invention can be used for therapeutic purposes.

[0290] In one aspect, an rAAV vector is provided for use as a medicament. In other aspects, an rAAV vector is provided for treating diseases caused by loss of gene function in patients. In some aspects, an rAAV vector is provided for use in a treatment method. In some aspects, the invention provides a method of using an rAAV vector to treat an individual suffering from a loss-of-function hereditary disease, the method comprising administering an effective amount of the rAAV vector to said individual. A “loss-of-function hereditary disease” refers to a hereditary disease in which gene mutations or other genetic defects result in a reduction or absence of the production of functional proteins, thereby leading to a disease phenotype. Therapies for treating such diseases are also known in the field as gene replacement therapy. Examples of such diseases include, but are not limited to, cystic fibrosis, sickle cell anemia, hemophilia, and Tay-Sachs disease. In one such aspect, as described below, 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.

[0291] In a further aspect, the present invention provides the use of rAAV carriers in the preparation or manufacture of pharmaceuticals. In one aspect, the pharmaceutical product is used to treat a loss-of-function genetic disease. In another aspect, a method of using the pharmaceutical product to treat a loss-of-function genetic disease includes administering an effective amount of the pharmaceutical product to an individual suffering from the loss-of-function genetic disease. In one such aspect, as described below, the method further includes administering an effective amount of at least one additional therapeutic agent to the individual.

[0292] In a further aspect, the present invention provides a method for treating loss-of-function inherited diseases. In one aspect, the method includes administering an effective amount of an rAAV vector to an individual suffering from such a loss-of-function inherited disease. In another aspect, as described below, the method further includes administering an effective amount of at least one additional therapeutic agent to the individual.

[0293] The individual according to any of the above aspects is preferably a person.

[0294] In a further aspect, the present invention provides pharmaceutical compositions comprising any of the rAAV carriers provided herein, for example, for use in any of the above-described treatment methods. In one aspect, the pharmaceutical composition comprises any of the rAAV carriers provided herein and a pharmaceutically acceptable carrier. In another aspect, the pharmaceutical composition comprises any of the rAAV carriers provided herein and at least one additional therapeutic agent, such as those described below.

[0295] The rAAV carrier of the present invention can be administered alone or in combination therapy. For example, such combination therapy includes administering the rAAV carrier of the present invention and administering at least one additional therapeutic agent (e.g., one, two, three, four, five, or six additional therapeutic agents).

[0296] The aforementioned combination therapies encompass both combined administration (where two or more therapeutic agents are included in the same or separate pharmaceutical compositions) and single administration. In the case of single administration, the rAAV carrier of the present invention may be administered before, simultaneously with, and / or after the administration of additional therapeutic agents or pharmaceutical agents. In one aspect, the administration of the rAAV carrier and the administration of additional therapeutic agents are performed within approximately one month of each other, or within approximately one week, two weeks, or three weeks, or within approximately one day, two days, three days, four days, five days, or six days. In one aspect, the rAAV carrier and the additional therapeutic agent are administered to the patient on the first day of treatment.

[0297] The rAAV carrier (and any other therapeutic agent) produced according to the invention can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, and, if desired, for local treatment or intralesional application. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Administration can be carried out by any suitable route, such as by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is transient or long-term. Various dosing schedules are considered herein, including but not limited to single or multiple administrations at various time points, bolus administration, and pulsatile infusion.

[0298] The rAAV carrier produced according to the invention will be formulated, administered, and applied in accordance with good medical practice. Factors to be considered in this context include the specific disease being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the disease, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to a practicing physician. The rAAV carrier is not mandatory but may optionally be formulated in conjunction with one or more formulations currently used for the prevention or treatment of the disease in question. The effective amount of these other formulations depends on the amount of rAAV carrier present in the pharmaceutical composition, the type of disease or treatment, and other factors discussed above.

[0299] For the prevention or treatment of disease, the appropriate dosage of the rAAV carrier produced according to the present invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of rAAV carrier, the severity and course of the disease, whether the rAAV carrier is administered for preventive or therapeutic purposes, the patient's clinical history, and the judgment of the attending physician. The rAAV carrier is appropriately administered to the patient once or in a series of treatments. The progress of this therapy can be easily monitored using routine techniques and assays.

[0300] In another aspect of the invention, an article is provided containing a substance that can be used to treat, prevent, and / or diagnose the aforementioned diseases. The article includes a container and a label or packaging insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous (IV) solution bags, etc. The container can be formed from a variety of materials such as glass or plastic. The container contains a composition that, on its own or in combination with another composition, is effective for treating, preventing, and / or diagnosing the condition, and the container may have a sterile inlet (e.g., the container may be an IV solution bag or vial with a stopper capable of being punctured by a hypodermic needle). At least one active agent in the composition is an rAAV carrier produced according to the invention. The label or packaging insert indicates that the composition is for treating the selected condition. Furthermore, the article may include (a) a first container containing the composition, wherein the composition comprises an rAAV carrier produced according to the invention; and (b) a second container containing the composition, wherein the composition contains additional cytotoxic agents or other therapeutic agents. The article in this aspect of the invention may also include a packaging insert indicating that the composition is for treating a specific condition. Alternatively or additionally, the article may further comprise a second (or third) container containing pharmaceutical buffers, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and glucose solution. It may further include other materials desired from a commercial and user perspective, including additional buffers, diluents, filters, needles, and syringes.

[0301] Specific embodiments of the invention are described in the following statements:

[0302] 1. A recombinant transcription unit comprising a nucleotide sequence encoding a polypeptide, wherein the nucleotide sequence is operatively linked to a recombinant polyadenylation signal sequence, wherein the recombinant polyadenylation signal sequence has a sequence length of less than 100 nucleotides, and wherein 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 the polypeptide in 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 sequence identity.

[0303] 2. The recombinant transcription unit according to Example 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 the nucleotide 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.

[0304] 3. A recombinant transcription unit comprising a nucleotide sequence encoding a polypeptide, wherein the nucleotide sequence is operatively linked to a recombinant polyadenylation signal sequence, wherein the recombinant polyadenylation signal sequence comprises or is composed of a nucleotide sequence selected from or consisting of 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.

[0305] 4. The recombinant transcription unit according to any one of Examples 1 to 3, wherein the recombinant polyadenylation signal sequence comprises or consists of a nucleotide sequence selected from or composed of the group consisting of SEQ ID NO:6, SEQ ID NO:9 and SEQ ID NO:12.

[0306] 5. A recombinant nucleic acid comprising:

[0307] (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

[0308] (b) A second recombinant transcription unit comprising a second nucleotide sequence encoding a second polypeptide operably linked to a second recombinant polyadenylation signal sequence.

[0309] The first recombinant polyadenylation signal sequence and the second recombinant polyadenylation signal sequence have less than 70%, 65%, 60%, 55%, 50%, 45%, or 40% sequence identity.

[0310] 6. The recombinant nucleic acid according to Example 5, further comprising:

[0311] (c) A third recombinant transcription unit comprising a third nucleotide sequence encoding a third polypeptide that is operatively linked to a third recombinant polyadenylation signal sequence.

[0312] The first and second recombinant polyadenylation signal sequences individually share less than 70%, 65%, 60%, 55%, 50%, 45%, or 40% sequence identity with the third recombinant polyadenylation signal sequence.

[0313] 7. The recombinant nucleic acid according to Example 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.

[0314] 8. The recombinant nucleic acid according to Examples 5 to 7, wherein the first recombinant polyadenylation signal sequence, the second recombinant polyadenylation signal sequence, and, if present, the third recombinant polyadenylation signal sequence, cannot participate in DNA strand exchange to form a recombinant intermediate.

[0315] 9. The recombinant nucleic acid according to any one of Examples 5 to 8, wherein recombination events between the nucleic acid containing the first recombinant polyadenylation signal sequence and the nucleic acid containing the second recombinant polyadenylation signal sequence are reduced or prevented.

[0316] 10. The recombinant nucleic acid according to any one of Examples 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.

[0317] 11. The recombinant nucleic acid according to any one of Examples 5 to 10, wherein the first polypeptide, the second polypeptide, and, if present, the third polypeptide are expressed in eukaryotic cells.

[0318] 12. The recombinant nucleic acid according to any one of Examples 5 to 11, wherein the first recombinant transcription unit is the recombinant transcription unit according to any one of Examples 1 to 4, and wherein the second recombinant transcription unit is the recombinant transcription unit according to any one of Examples 1 to 4, and wherein, if present, the third recombinant transcription unit is the recombinant transcription unit according to any one of Examples 1 to 4.

[0319] 13. The recombinant nucleic acid according to any one of Examples 5 to 12, wherein

[0320] (a) The first recombinant transcription unit further comprises a first promoter operatively linked to a nucleotide sequence encoding a first polypeptide, and

[0321] (b) The second recombinant transcription unit further comprises a second promoter operatively linked to a nucleotide sequence encoding a second polypeptide.

[0322] The first promoter and the second promoter have sequence identity of less than 70%, 65%, 60%, 55%, 50%, 45%, or 40%.

[0323] 14. The recombinant nucleic acid according to any one of Examples 6 to 13, wherein:

[0324] (c) If the first recombinant transcription unit further comprises a first promoter operatively linked to a nucleotide sequence encoding a first polypeptide,

[0325] The first promoter and the second promoter have sequence identity with the third promoter of less than 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 65%, or 60%.

[0326] 15. The recombinant nucleic acid according to Example 13 or 14, wherein

[0327] (i) The first promoter, the second promoter, and, if present, the third promoter, are active in eukaryotic cells.

[0328] (ii) The first promoter drives the expression of the first polypeptide.

[0329] (iii) The second promoter drives the expression of the second polypeptide.

[0330] (iv) The third promoter drives the expression of the third polypeptide, and / or

[0331] (v) The first promoter, the second promoter, and, if present, the third promoter drive the expression of the first polypeptide, the second polypeptide, and, if present, the third polypeptide, respectively.

[0332] 16. The recombinant nucleic acid according to any one of Examples 13 to 15, wherein the first promoter, the second promoter, and, if present, the third promoter are individually selected from the group consisting of the hPGK1 promoter, the CMV promoter, and the hEF1α promoter.

[0333] 17. The method according to any one of Examples 5 to 16, wherein the recombinant nucleic acid comprises at least one vector.

[0334] 18. The recombinant nucleic acid according to any one of Examples 5 to 17, wherein the recombinant nucleic acid comprises: a first vector comprising a first recombinant transcription unit; a second vector comprising a second recombinant transcription unit; and a third vector comprising a third recombinant transcription unit if a third recombinant transcription unit is present.

[0335] 19. The recombinant nucleic acid according to any one of Examples 17 or 18, wherein at least one vector comprises an optional marker operatively linked to a first recombinant transcription unit, a second recombinant transcription unit, or, if present, a third recombinant transcription unit.

[0336] 20. The recombinant nucleic acid according to Example 19, wherein the selectable marker is selected from hygromycin selectable marker, neomycin selectable marker, G418 selectable marker, dihydrofolate reductase (DHFR), thymidine kinase, glutamine synthase, asparagine synthase, tryptophan synthase, histidine dehydrogenase, and nucleic acids conferring resistance to puromycin, bleomycin, humulin, chloramphenicol, bleomycin, and mycophenolic acid.

[0337] 21. The recombinant nucleic acid according to Examples 17 to 20, wherein the first vector, the second vector and / or, if present, the third vector, contains a bacterial origin of replication, particularly the pUC19 origin of replication.

[0338] 22. A host cell comprising a recombinant transcription unit according to any one of Examples 1 to 4 and / or a recombinant nucleic acid according to any one of Examples 5 to 21.

[0339] 23. The host cell according to Example 22 is a eukaryotic host cell.

[0340] 24. The host cell according to Example 22 or 23 is selected from the group consisting of CHO, BHK, HEK and Sp2 / O.

[0341] 25. A recombinant viral vector comprising a vector genome, wherein the vector genome is comprised in a 5' to 3' sequence:

[0342] (i) 5' ITR sequence,

[0343] (ii) Startup subsequence

[0344] (iii) The sequence encoding the polypeptide.

[0345] (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

[0346] (v)3' ITR sequence.

[0347] 26. The recombinant viral vector according to Example 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.

[0348] 27. The recombinant viral vector according to Example 25 or 26, wherein the recombinant viral vector is selected from the group consisting of retroviral vectors, adenovirus vectors, helper-dependent adenovirus vectors, heterozygous adenovirus vectors, herpes simplex virus vectors, lentiviral vectors, poxvirus vectors, Epstein-Barr virus vectors, vaccinia virus vectors, human cytomegalovirus vectors, lentiviral vectors, adenovirus vectors or adeno-associated virus (AAV) vectors, or recombinant variants derived therefrom.

[0349] 28. The recombinant viral vector according to any one of Examples 25 to 27, wherein the recombinant viral vector is a recombinant adeno-associated virus (rAAV) vector.

[0350] 29. The rAAV according to Example 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 capsids or variant capsids derived therefrom.

[0351] 30. A method for generating a target polypeptide, the method comprising the following steps

[0352] (a) Providing a host cell according to any one of Examples 22 to 24,

[0353] (b) Incubate host cells under conditions suitable for peptide expression.

[0354] (c) Recover the target peptide from the cell culture.

[0355] 31. A method for generating a target polypeptide, the method comprising the following steps

[0356] (a) Providing a host cell comprising the recombinant nucleic acid according to any one of Examples 4 to 21, wherein the target polypeptide is a first polypeptide, and wherein the second polypeptide and, if present, a third polypeptide are necessary for or improve the production of the target polypeptide.

[0357] (b) Incubate host cells under conditions suitable for the expression of the first polypeptide, the second polypeptide, and, if present, a third polypeptide.

[0358] (c) Recovery of the target peptide from cell cultures, and optionally

[0359] (d) Formulate the recovered target peptide for therapeutic use.

[0360] 32. A method for generating a recombinant adeno-associated virus (rAAV) vector, the method comprising the following steps

[0361] (a) Providing a host cell comprising the recombinant nucleic acid according to any one of Examples 4 to 21, wherein a first polynucleotide sequence encodes a therapeutic payload, wherein a second nucleotide sequence encodes the viral vector rep and cap proteins, and wherein a third nucleotide sequence encodes the E4, E2a, and VA proteins.

[0362] (b) Incubate host cells under conditions suitable for generating the recombinant rAAV vector, and

[0363] (c) Recovery of viral vectors from cell cultures, and optionally

[0364] (d) Formulate the recovered target peptide for therapeutic use.

[0365] 33. The method according to any one of Examples 30 to 32, wherein the host cell is selected from the group consisting of CHO cells, BHK cells, HEK cells and Sp2 / O cells.

[0366] 34. The method according to Example 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.

[0367] 35. Use of a recombinant transcription unit for recombinant production of a target polypeptide, wherein the recombinant transcription unit is defined according to any one of Examples 1 to 4.

[0368] 35. Use of a recombinant nucleic acid for recombinant production of a target polypeptide, wherein the recombinant nucleic acid is defined according to any one of Examples 5 to 21.

[0369] 36. The invention as described above with reference to the examples and figures contained herein.

[0370] Exemplary sequence

[0371]

[0372] ***

[0373] This disclosure includes combinations of the described aspects and preferred features, unless such combination is obviously not permitted or explicitly avoided.

[0374] Aspects and embodiments of this disclosure will now be illustrated by way of example with reference to the accompanying drawings. Other aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated by reference.

[0375] Throughout the specification (including the following claims), unless the context otherwise requires, the word “comprising” and variations such as “including” and “containing” should be understood to imply inclusion of the stated integer or step or group of integers or steps, but not to exclude any other integer or step or group of integers or steps.

[0376] It should be noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context explicitly specifies otherwise. A range herein may be expressed as “about” a particular value and / or to “about” another particular value. When expressing such a range, another embodiment includes from one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation, the use of the antecedent “about” will be understood to form another embodiment of a particular value.

[0377] When this paper discloses the nucleic acid sequence, its reverse complementary sequence is also explicitly considered.

[0378] The methods described herein are preferably performed in vitro. The term "in vitro" is intended to cover procedures performed in cultures using cells, while the term "in vivo" is intended to cover procedures performed using / on intact multicellular organisms.

[0379] Example

[0380] The following are examples of the methods and compositions of the present invention. It should be understood that various other embodiments may be practiced given the general description provided above.

[0381] Example 1

[0382] Materials and methods

[0383] 1.1 Gene Synthesis

[0384] The desired gene fragments and plasmids need to be synthesized by GenScript Biotech (Rijswijk, Netherlands).

[0385] 1.2 Cell culture and transfection of human embryonic kidney cells (HEK293T)

[0386] HEK293T cells were cultured in DMEM (high glucose, GlutaMAX, pyruvate, 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 passaged using 0.25% trypsin-EDTA (Gibco, catalog number 25200).

[0387] For transient transfection of HEK293T cells, 2500 cells were seeded into 384-well plates at 20 μL per well one day prior to transfection. Each well was then transiently transfected with a 5 μL transfection mixture consisting of 25 ng of plasmid DNA infused with 0.05 μL of Lipofectamine 2000 (Invitorgen, catalog number 11668019) in Opti-MEM reduced serum medium (Gibco, catalog number 31985) at room temperature for 20 min. All reporter plasmids used to assess polyadenylation signaling were transfected in equimolar amounts, and the total amount of transfected plasmid for each experimental condition was normalized to 25 ng using mimic plasmids lacking active transcription and open reading frames.

[0388] 1.3 Quantitative Production of NanoLuc Luciferase (Nluc)

[0389] Total Nlucose production was quantified using a Nano-Glo luciferase assay system (Promega, catalog number N1110) by adding 25 μL of 2X luciferase assay solution (1 Vol Nano-Glo luciferase assay substrate mixed with 50 Vol assay buffer) to each well of a 384-well plate. 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.

[0390] Example 2

[0391] A transient transfection reporter plasmid was assembled using DNA sequences encoding a constitutive promoter (Prom.), enhanced green fluorescent protein (EGFP), a P2A self-cleaving peptide sequence, NanoLuc luciferase (Nluc), a PEST protein degradation signal, and a 3' untranslated region (3'UTR) to assess the ability of polyadenylation (polyA) signaling to support high expression levels through efficient transcription termination and polyadenylation (Figure 1).

[0392] Two copies (2x sNRP-1; McFarland et al. (2006)) of the standard BGH polyadenylation signal sequence and the short sNRP-1 polyadenylation signal sequence were inserted into the reporter plasmid located directly downstream of the 3' UTR. Figure 2A To assess the relative transcription termination efficiency and ability of recombinant polyadenylation signal sequences to support high protein expression levels, HEK293T cells were transiently transfected with the corresponding reporter plasmids for 24 hours, and total Nluc expression levels were then measured. Figure 2B shows the relative Nluc expression levels of the two reporter plasmids, with results normalized to the mean luminescence values ​​of cells transfected with the BGH-encoded reporter plasmid. Notably, the short 2x sNRP-1 polyA-encoded construct showed <25% expression compared to the BGH-encoded construct, highlighting the reliability of short polyadenylation signal sequences known in the art for supporting high expression levels of target genes.

[0393] Example 3

[0394] To establish a set of short recombinant polyadenylation (PA) signal sequences that can support high expression levels while exhibiting high sequence heterogeneity for use in multigene expression vectors without recombination risk, a 95-nucleotide (nt) recombinant PAS signal sequence design was created. This design consists of core elements of the PAS signal sequence derived from the synthetic rabbit β-globulin PAS signal sequence defined by Levitt et al. (Levitt et al. (1989)), including the PAS signal and two GU / U-rich downstream sequence elements (DSE) regions. Furthermore, 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).

[0395] Based on the above polyA design, an initial set of four recombinant polyadenylation signal sequences (polyA-1.1, -2.1, -3.1, and -4.1) was designed. Each of the four recombinant polyadenylation signal sequences was then rationally modified by extending the usage region to 46 nt, and designed to include unique primer annealing sites with a Tm of 70-72 °C, compatible with Gibson Assembly. Furthermore, small nt modifications were introduced, focusing on the use of the variable region between the PSA and DSE regions, to increase heterogeneity among polyA sequences or reduce strong secondary RNA structures within polyA sequences. Twelve recombinant polyadenylation signal sequences were selected and introduced into the aforementioned reporter plasmid (Figure 4A), and transiently detected in HEK293T. Figure 4B shows the relative Nluc expression levels from the reporter plasmids encoding the 12 recombinant polyadenylation signal sequences 24 hours post-transfection, with results normalized to the average luminescence values ​​for all transfection conditions. Three (polyA-2.3, -3.3, and -4.3) were selected from the set of recombinant polyadenylation signal sequences tested for further characterization because they were able to maintain higher relative expression levels and had <50% sequence similarity to each other.

[0396] Example 4

[0397] To benchmark against three selected recombinant polyadenylation signal sequences, the rabbit β-globulin polyadenylation signal sequence defined by Levitt et al. (Levitt et al. (1989)) was incorporated into a reporter plasmid (Fig. 5A) and transiently tested in HEK293T. Fig. 5B shows the relative Nluc expression levels of Levitt et al. polyA-encoding plasmids and the three synthetic polyA-encoding plasmids 24 hours post-transfection, with results normalized to the mean luminescence values ​​of cells transfected with the Levitt et al. polyA-encoding reporter plasmid. Notably, the Nluc expression levels of all three synthetic polyA-encoding constructs were more than twice that of the Levitt et al. rabbit β-globulin polyadenylation signal sequence.

[0398] Example 5

[0399] To benchmark the three selected recombinant polyadenylation (PA) signal sequences against known larger PA signal sequences, hGH polyA and SV40 polyA were introduced into the reporter gene construct, and plasmids containing the recombinant PA signal sequences were tested using reporter plasmids containing BGH, as described above (Figure 6A). To assess the relative transcription termination efficiency and ability to support high protein expression levels of the recombinant PA signal sequences, HEK293T cells were transiently transfected with the corresponding reporter plasmids for 24 hours, and total Nluc expression levels were then measured. Figure 6B shows the relative Nluc expression levels from the reporter plasmids. The results were normalized to the mean luminescence values ​​of cells transfected with BGH-encoded reporter plasmids. Notably, the recombinant PA signal sequences supported expression levels comparable to or higher than those of known larger PA signal sequences.

[0400] Example 6

[0401] To assess how the recombinant polyadenylation signal sequence is affected by the upstream 3'UTR sequence composition, three different de novo 3'UTR sequences sharing <50% paired sequence identity and <30% identical sequence were introduced into the recombinant polyadenylation signal sequence encoding the reporter plasmid (Figure 7A). As shown in Figure 7B, all constructs were transiently tested in HEK293T to describe the relative Nluc expression levels from the reporter plasmid 24 hours post-transfection, and the results were normalized to the average luminescence values ​​for all transfection conditions. Notably, the recombinant polyadenylation signal sequence showed minimal influence from the three different upstream 3'UTR sequences.

[0402] Example 7

[0403] To assess how the recombinant polyadenylation signal sequence is affected by the strength of the promoter used and the resulting expression level, three constitutive promoters of different strengths, including hPGK1, CMV, and hEF1α, were introduced into the recombinant polyadenylation signal sequence encoding their respective reporter plasmids (Figure 8A). All constructs were briefly tested in HEK293T, and the relative Nluc expression level from the reporter plasmid was quantified 24 hours post-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 average luminescence values ​​for all transfection conditions in the corresponding figures. Notably, the binding of the recombinant polyadenylation signal sequence to different constitutive promoters supports robust expression at different levels.

[0404] * * *

Claims

1. A recombinant transcription unit comprising a nucleotide sequence encoding a polypeptide, wherein the nucleotide sequence is operatively linked to a recombinant polyadenylation signal sequence, wherein the recombinant polyadenylation signal sequence has a sequence length of less than 100 nucleotides, and wherein 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 compared to the expression level of the polypeptide in 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 sequence identity.

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 the nucleotide 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.

3. A recombinant transcription unit comprising a nucleotide sequence encoding a polypeptide, wherein the nucleotide sequence is operatively linked to a recombinant polyadenylation signal sequence, wherein the recombinant polyadenylation signal sequence comprises or consists of a nucleotide 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.

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 or composed of the group consisting of SEQ ID NO:6, SEQ ID NO:9 and SEQ ID NO:

12.

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

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

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, if present, the third recombinant polyadenylation signal sequence, cannot participate in DNA strand exchange to form a recombinant intermediate.

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

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

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

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

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 comprises, in a 5' to 3' sequence: (i) 5' ITR sequence, (ii) Starter sequence, (iii) The sequence encoding the 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) 3' ITR sequence.

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

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

17. A method for generating a target polypeptide, the method comprising the following steps: (a) Providing a host cell according to claim 12, (b) Incubate the host cells under conditions suitable for expressing the polypeptide. (c) The target polypeptide is recovered from the cell culture.

18. A method for generating a target polypeptide, the method comprising the following steps: (a) Providing a host cell comprising the recombinant nucleic acid according to any one of claims 5 to 11, wherein the target polypeptide is a first polypeptide, and wherein the second polypeptide and, if present, a third polypeptide are necessary for or improve the production of the target polypeptide. (b) Incubate the host cells under conditions suitable for the expression of the first polypeptide, the second polypeptide, and, if present, the third polypeptide. (c) Recover the target polypeptide from the cell culture, and optionally... (d) Formulate the recovered target peptide for therapeutic use.

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

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