Cloning / expression vector for non-b DNA sequence

Abstract

Provided is a vector. The vector comprises: i) a replication origin; ii) a first terminator; iii) a) a target DNA sequence, comprising a DNA sequence capable of forming a non-B DNA conformation, or b) a target DNA expression cassette, comprising a promoter and a target DNA sequence, wherein the target DNA sequence comprises a DNA sequence capable of forming a non-B DNA conformation; iv) a second terminator; and v) a selectable marker and a promoter thereof, wherein in a replication direction, the replication origin is at least 150 bp apart from the DNA sequence capable of forming the non-B DNA conformation, the first terminator and the second terminator are respectively located upstream and downstream of the target DNA sequence in a), or the first terminator and the second terminator are respectively located upstream and downstream of the target DNA expression cassette in b). The provided vector can improve the amplification and / or expression (transcription) stability of poly(A) tail and ITR sequences.

Description

Cloning / expression vectors for non-B-type DNA sequences

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202311099203.6 filed on August 29, 2023, the entire contents of which are incorporated herein by reference. Field of the Invention

[0003] The present application relates to a cloning vector or expression vector that can be used for, for example, a sequence that can form a non-B-form DNA conformation, as well as the construction and use of these vectors. Background Art

[0004] mRNA vaccines are nucleic acid preparations prepared by transcribing exogenous target genes or direct synthesis. They can be introduced into the body's cells through a specific delivery system and express the target protein, thereby stimulating the body to produce a specific immunological response and provide immune protection.

[0005] The bulk of a mature mRNA is the coding region, flanked by noncoding regions (UTRs) on both the 5' upstream and 3' downstream sides. Eukaryotic mRNA molecules also contain a 5' cap and a 3' poly(A) tail. The poly(A) tail plays a crucial role in maintaining mRNA stability, regulating translation efficiency, and maintaining mRNA transport. Currently, in vitro transcription (IVT) is the most efficient method for bulk mRNA synthesis. Within IVT, there are two main approaches for poly(A) tailing. The first involves enzymatic synthesis. After mRNA is transcribed from a linear DNA template, poly(A) polymerase is added to catalyze the addition of AMP, converted from ATP, to the 3' end of the single-stranded RNA, forming the poly(A) tail. This method has the advantage of being simple to use, as it does not require a poly(A) sequence on the template DNA. However, it suffers from the limitation of unstable tail length. The second method is co-transcription, which involves direct transcription of the poly(A) tail coding sequence already present on the template plasmid DNA or PCR product. This approach has the advantage of not requiring the use of poly(A) polymerase, reducing process steps and costs, and maintaining a relatively stable length of the transcribed tail. However, during upstream plasmid preparation, it is crucial to minimize the loss of the poly(A) tail coding sequence during culture, which is crucial for providing high-yield, high-quality, and uniform plasmid templates.

[0006] Large-scale production of plasmids is typically performed via Escherichia coli fermentation. During amplification, plasmids carrying long poly(A) tail coding sequences are inherently unstable and prone to loss during replication. To improve the stability of the poly(A) tail coding sequence on plasmids, some scientists have employed segmented poly(A) tail sequences. Because each poly(A) binding protein, PABP, only binds approximately 30 A residues, a small number of A residues between the two binding proteins acts as a spacer sequence. Replacing the spacer sequence with a non-A base reduces the probability of recombination within the poly(A) tail coding sequence. Furthermore, the length and base number of the spacer sequence can be optimized to further reduce the risk of poly(A) tail loss. However, even with this optimization, the risk of A loss still exists, and segmented production of poly(A) tail plasmids can negatively impact downstream protein expression. Furthermore, low-temperature fermentation (30°C) has been reported to improve the integrity of the poly(A) tail coding sequence on plasmids. However, low-temperature cultivation slows bacterial growth, reduces plasmid copy number, significantly reduces yield, and prolongs production cycles, making plasmid production difficult. Currently common commercial vectors used for mRNA vaccine production, such as pVAX1, also have the problems of low plasmid copy number, low yield and impure plasmid Poly (A) tail sequence in actual production.

[0007] Therefore, developing more stable poly(A) tail cloning vectors that can reduce the risk of poly(A) tail sequence deletion or impurity without affecting plasmid yield may become a new research direction. However, there are currently few reports on the effects of vectors and their components on poly(A) tail sequence stability.

[0008] Similarly, the inverted terminal repeats (ITRs) of adeno-associated virus (AAV) also have the problem of unstable amplification in the vector. ITRs are present at both ends of the AAV genome, each forming a special self-complementary T-shaped palindromic hairpin structure, and have a high GC content, which is prone to mutation or recombination loss during bacterial reproduction. The integrity of ITRs is very important for the efficient replication and packaging of recombinant AAV (rAAV), and the optimal ITR deletion is less than 20% (Flotte, TR, and BJ Carter. (1995) Adeno-associated virus vectors for gene therapy, Gene therapy 2 (6): 357-362). Single-molecule real-time sequencing, which comprehensively analyzes the rAAV genome as a single complete molecule, found that in the HEK293 three-plasmid packaging system, the deletion of ITRs is correlated with the packaging of non-vector sequences. The impact of ITR deletion on rAAV virus packaging and the application of rAAV in gene therapy is complex and can produce unexpected biological consequences through vector-host interactions. If incomplete ITR sequences are used in the preclinical stage, the reproducibility of experiments within and across laboratories will be reduced, and may lead to experimental duplication when clinical trials using complete ITR sequences, delaying the translation into clinical applications.

[0009] Currently, there is no effective method to prevent ITR deletion during rAAV-ITR plasmid construction and E. coli amplification. Commercially available recombination-deficient strains such as JC8111, SURE2, Stabl3, and XL10-Gold are often used to construct and amplify ITR-containing plasmids, but this is not always effective.

[0010] The citation of any document in this application does not constitute an admission that these documents are prior art to this application.

[0011] Summary of the Invention

[0012] The inventors of this application have experimentally discovered that by i) extending the distance between the replication origin and the poly(A) tail coding sequence, ITR sequence, and other sequences that are prone to mutation and deletion during replication; ii) adding terminators to the poly(A) tail coding sequence, ITR, and other sequences that are prone to mutation and deletion during replication, or to the upstream and downstream of their expression cassettes; and / or iii) destroying the original lac promoter functional region in the vector, the amplification and / or expression (transcription) stability of these sequences that are prone to deletion or mutation can be significantly improved. In addition, the above-mentioned changes to the vector can also increase the yield of the vector, especially the yield (such as packaging titer) of the recombinant adeno-associated virus (rAAV).

[0013] Thus, in a first aspect, the present application provides a vector, which may comprise:

[0014] i) origin of replication, and

[0015] ii) a) a target DNA expression cassette comprising a promoter and a target DNA sequence, or b) a target DNA sequence.

[0016] The target DNA sequence in the target DNA expression cassette of a) may include a DNA sequence capable of forming a non-B-form DNA configuration. The target DNA sequence in the target DNA expression cassette of a) may include a DNA sequence capable of forming a non-B-form DNA configuration and a cloning site. The cloning site may be a multiple cloning site.

[0017] The target DNA sequence of b) may include a DNA sequence capable of forming a non-B-form DNA configuration. The target DNA sequence of b) may include a DNA sequence capable of forming a non-B-form DNA configuration and a cloning site. The cloning site may be a multiple cloning site.

[0018] The DNA sequence capable of forming a non-B-form DNA configuration can be a forward repeat sequence, an inverted repeat sequence, or a single purine-single pyrimidine sequence, including but not limited to polyadenine (poly(A)), polyguanine (poly(G)), polycytosine (poly(C)), or polythymine (poly(T)), shRNA coding sequence, and ITR sequence. The forward repeat sequence can be a poly(A) tail coding sequence. The single purine-single pyrimidine sequence can be a poly(A) tail coding sequence. The inverted repeat sequence can be an ITR or a sequence derived therefrom. In some embodiments, the DNA sequence capable of forming a non-B-form DNA configuration can be a poly(A) coding sequence.

[0019] The replication origin may be separated from a) the DNA sequence capable of forming a non-B-form DNA configuration in the target DNA expression cassette, or b) the DNA sequence capable of forming a non-B-form DNA configuration in the target DNA sequence by at least 150 bp, 200 bp, 300 bp, 500 bp, or 700 bp in the direction of replication. In some embodiments, the replication origin may be separated from a) the DNA sequence capable of forming a non-B-form DNA configuration in the target DNA expression cassette, or b) the DNA sequence capable of forming a non-B-form DNA configuration in the target DNA sequence by at least 300 bp in the direction of replication. In some embodiments, the replication origin may be separated from a) the DNA sequence capable of forming a non-B-form DNA configuration in the target DNA expression cassette, or b) the DNA sequence capable of forming a non-B-form DNA configuration in the target DNA sequence by at least 500 bp in the direction of replication. In some embodiments, the replication origin may be separated from a) the DNA sequence capable of forming a non-B-form DNA configuration in the target DNA expression cassette, or b) the DNA sequence capable of forming a non-B-form DNA configuration in the target DNA sequence by at least 700 bp in the direction of replication.

[0020] The origin of replication can enable the vector to replicate in a suitable host cell. Suitable host cells can be prokaryotes such as Escherichia coli, or eukaryotic cells. The origin of replication can be selected from the group consisting of the pMB1 origin of replication, the pBR322 origin of replication, the ColE1 origin of replication, the R6K origin of replication, the p15A origin of replication, the pSC101 origin of replication, the pUC origin of replication, the pET origin of replication, and derivatives of these origins of replication. These derivatives of the origin of replication can also enable the vector to replicate in a suitable host cell. In some embodiments, the origin of replication can be the pUC origin of replication.

[0021] The vector may further comprise a second origin of replication, which may be separated in the direction of replication by at least 150 bp, 200 bp, 300 bp, 500 bp, or 700 bp from the DNA sequence capable of forming a non-B-form DNA configuration in a) the target DNA expression cassette or the DNA sequence capable of forming a non-B-form DNA configuration in b) the target DNA sequence. This second origin of replication may enable the vector to replicate in a suitable host cell, which may be a prokaryotic cell, such as Escherichia coli, or a eukaryotic cell.

[0022] Depending on the type of the replication origin, the vector may also contain corresponding Rep protein expression elements, including a Rep protein coding sequence and a corresponding promoter.

[0023] The vector may further comprise a first terminator and a second terminator, wherein the first terminator and the second terminator are respectively located upstream and downstream of the target DNA expression cassette of a), and are configured to terminate transcription outside the target DNA expression cassette of a) at the upstream and downstream of the target DNA expression cassette of a); or the first terminator and the second terminator are respectively located upstream and downstream of the target DNA sequence of b), and are configured to terminate transcription outside the target DNA sequence of b) at the upstream and downstream of the target DNA sequence of b). The first terminator may be a forward terminator, a bidirectional terminator, or a reverse terminator. The second terminator may be a forward terminator, a bidirectional terminator, or a reverse terminator. In some embodiments, the first terminator and the second terminator may both be bidirectional terminators. The first terminator and the second terminator may be independently selected from rrnB T1 terminator, rrnB T2 terminator, lambda t0 terminator, TonB terminator, Amp terminator, T7 terminator, and CYC1 terminator. In some embodiments, the first terminator may be rrnB T1 terminator and rrnB T2 terminator. In some embodiments, the second terminator can be a lambda t0 terminator.

[0024] The vector can also include a screening marker. The screening marker can be an antibiotic resistance gene, such as an ampicillin resistance gene, a tetracycline resistance gene, a chloramphenicol resistance gene, a kanamycin resistance gene or a streptomycin resistance gene. In particular, the screening marker is not present in the genome of the host cell to be transformed / transfected by the vector. In some embodiments, the screening marker is a kanamycin resistance gene. The vector can also include a promoter for the expression of the screening marker upstream of the screening marker.

[0025] The vector may be a circular vector, a non-viral vector, or a plasmid vector.

[0026] The vector does not contain the lac promoter capable of initiating transcription.

[0027] The target DNA expression cassette of a) may contain a recognition site for an IIS type restriction endonuclease (IIS enzyme). The target DNA sequence of b) may also contain a recognition site for an IIS enzyme. Except for the target DNA expression cassette of a) or the target DNA sequence of b), the vector does not contain the same IIS enzyme recognition site as that in the target DNA expression cassette of a) and the target DNA sequence of b).

[0028] The target DNA expression cassette of a) can be an expression cassette suitable for expressing the target DNA sequence in eukaryotic cells, and it can include components that allow the target DNA sequence to be expressed in eukaryotic cells. The promoter included in the target DNA expression cassette of a) can be a promoter that allows the target DNA sequence to be expressed in eukaryotic cells. The target DNA expression cassette of a) can also include an enhancer, a transcription terminator, etc., in particular, an enhancer and a transcription terminator that allow the target DNA sequence to be expressed in eukaryotic cells. The transcription terminator suitable for expressing the target DNA sequence in eukaryotic cells can be selected from SV40 poly(A), hGH poly(A), BGH poly(A), and rbGlob poly(A). In some embodiments, the target DNA expression cassette of a) can be an expression cassette suitable for expressing mRNA in eukaryotic cells, and it can include optional enhancers, promoters, 5'UTRs, open reading frames (ORFs), 3'UTRs, poly(A) tail coding sequences, and transcription terminators.

[0029] The target DNA expression cassette in a) may further comprise an enzyme cutting site for vector linearization.

[0030] In some embodiments, the vector may comprise:

[0031] i) the origin of replication,

[0032] ii) a) a target DNA expression cassette comprising a promoter, and a target DNA sequence, wherein the target DNA sequence comprises a DNA sequence capable of forming a non-B-form DNA configuration, or b) a target DNA sequence comprising a DNA sequence capable of forming a non-B-form DNA configuration, and

[0033] iii) a selection marker and a promoter for controlling the expression of the selection marker,

[0034] The replication origin may be spaced at least 500 bp, or in particular at least 700 bp away from a) the DNA sequence capable of forming a non-B-form DNA configuration in the target DNA expression cassette, or b) the DNA sequence capable of forming a non-B-form DNA configuration in the target DNA sequence in the replication direction.

[0035] In some embodiments, the vector may comprise:

[0036] i) the origin of replication,

[0037] ii) a first terminator,

[0038] iii) a) a target DNA expression cassette comprising a promoter, and a target DNA sequence, wherein the target DNA sequence comprises a DNA sequence capable of forming a non-B-form DNA conformation, or b) a target DNA sequence comprising a DNA sequence capable of forming a non-B-form DNA conformation,

[0039] iv) a second terminator, and

[0040] v) a selection marker and a promoter for controlling the expression of the selection marker,

[0041] The first terminator and the second terminator may be located upstream and downstream of the target DNA expression cassette in a), respectively, and configured so that transcription outside the target DNA expression cassette in a) in the vector terminates upstream and downstream of the target DNA expression cassette in a), or the first terminator and the second terminator may be located upstream and downstream of the target DNA sequence in b), respectively, and configured so that transcription outside the target DNA sequence in b) terminates upstream and downstream of the target DNA sequence in b).

[0042] The replication origin may be separated from a) the DNA sequence capable of forming a non-B-form DNA configuration in the target DNA expression cassette, or b) the DNA sequence capable of forming a non-B-form DNA configuration in the target DNA sequence by at least 500 bp or 700 bp in the replication direction.

[0043] In some embodiments, the vector may contain, in order, a pUC replication origin, rrnB T1 terminator and rrnB T2 terminator, a target DNA sequence, a lambda t0 terminator, and a kanamycin resistance element.

[0044] In some embodiments, the vector may contain, in order, a pUC replication origin, rnB T1 terminator and rrnB T2 terminator, a CMV enhancer, a CMV promoter, a target DNA sequence, a bGH poly(A) signal, a lambda t0 terminator, and a kanamycin resistance element.

[0045] In some embodiments, the vector may comprise, in order, a pUC replication origin, rrnB T1 terminator and rrnB T2 terminator, a CMV enhancer, a CMV promoter, an mRNA functional region sequence comprising a poly(A) tail coding sequence, a bGH poly(A) signal, a lambda t0 terminator, and a kanamycin resistance element. In some embodiments, the vector may comprise, in order, a pUC replication origin, rrnB T1 terminator and rrnB T2 terminator, a CMV enhancer, a CMV promoter, a 5'UTR, an open reading frame (ORF), a 3'UTR, a poly(A) tail coding sequence, a vector linearization site, a bGH poly(A) signal, a lambda t0 terminator, and a kanamycin resistance element.

[0046] Accordingly, the present application provides a carrier skeleton, which may include:

[0047] i) origin of replication, and

[0048] ii) a) a cloning site, b) a cloning site and a DNA sequence capable of forming a non-B-form DNA configuration, c) a DNA expression cassette comprising a promoter and a cloning site, or d) a DNA expression cassette comprising a promoter, a cloning site and a DNA sequence capable of forming a non-B-form DNA configuration.

[0049] The DNA sequence capable of forming a non-B-form DNA configuration can be a forward repeat sequence, an inverted repeat sequence, or a single purine-single pyrimidine sequence, including but not limited to polyadenine (poly(A)), polyguanine (poly(G)), polycytosine (poly(C)), or polythymine (poly(T)), shRNA coding sequence, and ITR sequence. The forward repeat sequence can be a poly(A) tail coding sequence. The single purine-single pyrimidine sequence can be a poly(A) tail coding sequence. The inverted repeat sequence can be an ITR or a sequence derived therefrom. In some embodiments, the DNA sequence capable of forming a non-B-form DNA configuration in the DNA expression cassette of d) can be a poly(A) tail coding sequence.

[0050] The origin of replication may be separated from the cloning site of a) , the DNA sequence capable of forming a non-B-form DNA configuration in b) , the cloning site in the DNA expression cassette, or the DNA sequence capable of forming a non-B-form DNA configuration in d) by at least 150 bp, 200 bp, 300 bp, 500 bp, or 700 bp in the direction of replication. In some embodiments, the origin of replication may be separated from the cloning site of a) , the DNA sequence capable of forming a non-B-form DNA configuration in b) , the cloning site in the DNA expression cassette, or the DNA sequence capable of forming a non-B-form DNA configuration in d) by at least 500 bp in the direction of replication. In some embodiments, the origin of replication may be separated from the cloning site of a) , the DNA sequence capable of forming a non-B-form DNA configuration in b) , the cloning site in the DNA expression cassette, or the DNA sequence capable of forming a non-B-form DNA configuration in d) by at least 700 bp in the direction of replication.

[0051] The origin of replication can enable the vector to replicate in a suitable host cell. Suitable host cells can be prokaryotes such as Escherichia coli, or eukaryotic cells. The origin of replication can be selected from the group consisting of the pMB1 origin of replication, the pBR322 origin of replication, the ColE1 origin of replication, the R6K origin of replication, the p15A origin of replication, the pSC101 origin of replication, the pUC origin of replication, the pET origin of replication, and derivatives of these origins of replication. These derivatives of the origin of replication can also enable the vector to replicate in a suitable host cell. In some embodiments, the origin of replication can be the pUC origin of replication.

[0052] The vector backbone may further comprise a second origin of replication, which may be spaced at least 150 bp, 200 bp, 300 bp, 500 bp, or 700 bp in the direction of replication from a) the cloning site, b) the DNA sequence capable of forming a non-B-form DNA configuration, c) the cloning site in the DNA expression cassette, or d) the DNA sequence capable of forming a non-B-form DNA configuration in the DNA expression cassette. This second origin of replication may allow the vector to replicate in a suitable host cell, which may be a prokaryotic cell, such as Escherichia coli, or a eukaryotic cell.

[0053] Depending on the type of the replication origin, the vector may also contain corresponding Rep protein expression elements, including a Rep protein coding sequence and a corresponding promoter.

[0054] The vector backbone may further comprise a first terminator and a second terminator, the first terminator and the second terminator being respectively located upstream and downstream of the cloning site of a), and being configured such that transcription outside the cloning site of a) in the vector terminates upstream and downstream of the cloning site of a); being respectively located upstream and downstream of the cloning site of b) and the DNA sequence capable of forming a non-B-type DNA configuration, and being configured such that transcription outside the cloning site of b) and the DNA sequence capable of forming a non-B-type DNA configuration in the vector terminates upstream and downstream of the cloning site of b) and the DNA sequence capable of forming a non-B-type DNA configuration; being respectively located upstream and downstream of the DNA expression cassette of c), and being configured such that transcription of the DNA expression cassette of c) in the vector terminates upstream and downstream of the DNA expression cassette of c); or being respectively located upstream and downstream of the DNA expression cassette of d), and being configured such that transcription outside the DNA expression cassette of d) in the vector terminates upstream and downstream of the DNA expression cassette of d). The first terminator may be a forward terminator, a bidirectional terminator, or a reverse terminator. The second terminator can be a forward terminator, a bidirectional terminator, or a reverse terminator. In some embodiments, the first terminator and the second terminator can both be bidirectional terminators. The first terminator and the second terminator can be independently selected from rrnB T1 terminator, rrnB T2 terminator, lambda t0 terminator, TonB terminator, Amp terminator, T7 terminator, and CYC1 terminator. In some embodiments, the first terminator can be rrnB T1 terminator and rrnB T2 terminator. In some embodiments, the second terminator can be lambda t0 terminator.

[0055] The cloning site in a) of the vector backbone can be a multiple cloning site. The cloning site in b) of the vector backbone can be a multiple cloning site. The cloning site in c) of the vector backbone DNA expression cassette can be a multiple cloning site. The cloning site in d) of the vector DNA expression cassette can be a multiple cloning site.

[0056] Any sequence can be inserted into the cloning site of a) of the vector backbone, in particular a target DNA sequence, or a promoter and a target DNA sequence, wherein the target DNA sequence can include a DNA sequence that can form a non-B-form DNA configuration. Any sequence can be inserted into the cloning site of b) of the vector backbone. Any sequence can be inserted into the cloning site of c) the DNA expression cassette of the vector backbone, in particular a target DNA sequence, wherein the target DNA sequence can include a DNA sequence that can form a non-B-form DNA configuration. Any sequence can be inserted into the cloning site of d) the DNA expression cassette of the vector backbone. In some embodiments, the DNA sequence that can form a non-B-form DNA configuration in d) the DNA expression cassette can be a poly (A) coding sequence, and an mRNA coding sequence can be inserted into the cloning site of d) the DNA expression cassette.

[0057] The vector backbone can also include a screening marker. The screening marker can be an antibiotic resistance gene, such as an ampicillin resistance gene, a tetracycline resistance gene, a chloramphenicol resistance gene, a kanamycin resistance gene or a streptomycin resistance gene. In particular, the screening marker is not present in the genome of the host cell to be transformed / transfected by the vector. In some embodiments, the screening marker is a kanamycin resistance gene. The vector can also include a promoter for the expression of the screening marker upstream of the screening marker.

[0058] The vector backbone may be a circular vector backbone, a non-viral vector backbone, or a plasmid vector backbone.

[0059] The vector backbone does not contain a lac promoter capable of initiating transcription.

[0060] The sequence to be added to the cloning site in a) , the DNA sequence capable of forming a non-B-form DNA configuration in b) , the sequence to be added to the cloning site of the DNA expression cassette in c) , or the DNA sequence capable of forming a non-B-form DNA configuration in the DNA expression cassette in d) may contain an IIS enzyme recognition site. In addition to the above sequences, the vector backbone does not contain the same IIS enzyme recognition sites as in a) to d).

[0061] The DNA expression cassettes of c) and d) can be expression cassettes suitable for expressing DNA sequences in eukaryotic cells, and can include components that enable expression of the DNA sequence added at the cloning site, or the DNA sequence added at the cloning site and a DNA sequence capable of forming a non-B-form DNA configuration, in eukaryotic cells. The promoters included in the DNA expression cassettes of c) and d) can be promoters that enable expression of the DNA sequence added at the cloning site, or the DNA sequence added at the cloning site and a DNA sequence capable of forming a non-B-form DNA configuration, in eukaryotic cells. The DNA expression cassettes of c) and d) can also include enhancers, transcription terminators, and the like, in particular enhancers and transcription terminators that enable expression of the DNA sequence added at the cloning site, or the DNA sequence added at the cloning site and a DNA sequence capable of forming a non-B-form DNA configuration, in eukaryotic cells. Transcription terminators suitable for expression of DNA sequences in eukaryotic cells can be selected from SV40 poly(A), hGH poly(A), BGH poly(A), and rbGlob poly(A). In some embodiments, the DNA expression cassette of d) can be an expression cassette suitable for expressing mRNA in eukaryotic cells, wherein the DNA sequence capable of forming a non-B-form DNA conformation can be a poly(A) tail encoding sequence, wherein the DNA expression cassette can include an optional enhancer, a promoter, a 5' UTR, a cloning site, a 3' UTR, a poly(A) tail encoding sequence, and a transcription terminator. The DNA expression cassette of c) or d) can also include a restriction enzyme site for vector linearization.

[0062] In some embodiments, the carrier backbone may comprise:

[0063] i) the origin of replication,

[0064] ii) a) a cloning site, b) a cloning site and a DNA sequence capable of forming a non-B-form DNA configuration, c) a DNA expression cassette comprising a promoter and a cloning site, or d) a DNA expression cassette comprising a promoter, a cloning site and a DNA sequence capable of forming a non-B-form DNA configuration, and

[0065] iii) a selection marker and a promoter for controlling the expression of the selection marker,

[0066] The replication origin may be separated from a) the cloning site, b) the DNA sequence capable of forming a non-B-form DNA configuration, c) the cloning site in the DNA expression cassette, or d) the DNA sequence capable of forming a non-B-form DNA configuration in the DNA expression cassette by at least 500 bp or 700 bp in the replication direction.

[0067] In some embodiments, the carrier backbone may comprise:

[0068] i) the origin of replication,

[0069] ii) a first terminator,

[0070] iii) a) a cloning site, b) a cloning site and a DNA sequence capable of forming a non-B-form DNA configuration, c) a DNA expression cassette comprising a promoter and a cloning site, or d) a DNA expression cassette comprising a promoter, a cloning site and a DNA sequence capable of forming a non-B-form DNA configuration,

[0071] iv) a second terminator, and

[0072] v) a selection marker and a promoter for controlling the expression of the selection marker,

[0073] The first terminator and the second terminator may be located upstream and downstream of the cloning site in a), respectively, and are configured such that transcription outside the cloning site in a) in the vector terminates upstream and downstream of the cloning site in a).

[0074] The first terminator and the second terminator can be located upstream and downstream of the cloning site b) and the DNA sequence capable of forming a non-B-form DNA configuration, respectively, and are arranged so that transcription outside the cloning site b) and the DNA sequence capable of forming a non-B-form DNA configuration in the vector is terminated upstream and downstream of the cloning site b) and the DNA sequence capable of forming a non-B-form DNA configuration.

[0075] The first terminator and the second terminator can be located upstream and downstream of the DNA expression cassette of c), respectively, and are configured so that transcription outside the DNA expression cassette of c) in the vector is terminated upstream and downstream of the DNA expression cassette of c), or

[0076] The first terminator and the second terminator may be located upstream and downstream of the DNA expression cassette of d), respectively, and configured such that transcription outside the DNA expression cassette of d) in the vector terminates upstream and downstream of the DNA expression cassette of d);

[0077] The replication origin may be separated from a) the cloning site, b) the DNA sequence capable of forming a non-B-form DNA configuration, c) the cloning site in the DNA expression cassette, or d) the DNA sequence capable of forming a non-B-form DNA configuration in the DNA expression cassette by at least 500 bp or 700 bp in the replication direction.

[0078] In some embodiments, the vector backbone may comprise, in order, a pUC replication origin, rrnB T1 terminator and rrnB T2 terminator, a cloning site, a lambda t0 terminator, and a kanamycin resistance element.

[0079] In some embodiments, the vector backbone may comprise, in order, a pUC replication origin, rrnB T1 terminator and rrnB T2 terminator, a cloning site, a target DNA sequence, a lambda t0 terminator, and a kanamycin resistance element.

[0080] In some embodiments, the vector backbone can contain, in order, a pUC replication origin, rrnB T1 terminator and rrnB T2 terminator, a CMV enhancer, a CMV promoter, a cloning site, a bGH poly(A) signal, a lambda t0 terminator, and a kanamycin resistance element.

[0081] In some embodiments, the vector backbone may comprise, in order, a pUC replication origin, rrnB T1 terminator and rrnB T2 terminator, a CMV enhancer, a CMV promoter, a cloning site, a poly (A) tail coding sequence, a vector linearization site, a bGH poly (A) signal, a lambda t0 terminator, and a kanamycin resistance element. In some embodiments, the vector backbone may comprise, in order, a pUC replication origin, rrnB T1 terminator and rrnB T2 terminator, a CMV enhancer, a CMV promoter, a 5'UTR, a cloning site, a 3'UTR, a poly (A) tail coding sequence, a vector linearization site, a bGH poly (A) signal, a lambda t0 terminator, and a kanamycin resistance element.

[0082] In a second aspect, the present application provides a vector, which may comprise:

[0083] i) origin of replication, and

[0084] ii) a) a target DNA expression cassette comprising, in sequence, a first ITR or a sequence derived therefrom, a promoter, a target DNA sequence, and a second ITR or a sequence derived therefrom, or b) a target DNA sequence group comprising, in sequence, a first ITR or a sequence derived therefrom, a target DNA sequence, and a second ITR or a sequence derived therefrom.

[0085] The origin of replication can be spaced at least 150bp, 200bp, 300bp, 500bp, 700bp, 800bp, 900bp, 1000bp, 1100bp, 1200bp, 1300bp, 1400bp, 1500bp, 1600bp, or 1700bp in the direction of replication from the target DNA expression cassette of a) or the target DNA sequence group of b). In some embodiments, the origin of replication can be spaced at least 300bp in the direction of replication from the target DNA expression cassette of a) or the target DNA sequence group of b). In some embodiments, the origin of replication can be spaced at least 500bp in the direction of replication from the target DNA expression cassette of a) or the target DNA sequence group of b). In some embodiments, the origin of replication can be spaced at least 700bp in the direction of replication from the target DNA expression cassette of a) or the target DNA sequence group of b).

[0086] The origin of replication can enable the vector to replicate in a suitable host cell. Suitable host cells can be prokaryotes such as Escherichia coli, or eukaryotic cells. The origin of replication can be selected from the group consisting of the pMB1 origin of replication, the pBR322 origin of replication, the ColE1 origin of replication, the R6K origin of replication, the p15A origin of replication, the pSC101 origin of replication, the pUC origin of replication, the pET origin of replication, and derivatives of these origins of replication. These derivatives of the origin of replication can also enable the vector to replicate in a suitable host cell. In some embodiments, the origin of replication can be the pUC origin of replication.

[0087] The vector may further comprise a second origin of replication, which may be spaced at least 150 bp, 200 bp, 300 bp, 500 bp, 700 bp, 800 bp, 900 bp, 1000 bp, 1100 bp, 1200 bp, 1300 bp, 1400 bp, 1500 bp, 1600 bp, or 1700 bp in the direction of replication from a) the DNA expression cassette or b) the target DNA sequence group. This second origin of replication may allow the vector to replicate in a suitable host cell, which may be a prokaryotic cell such as Escherichia coli, or a eukaryotic cell.

[0088] Depending on the type of the replication origin, the vector may also contain corresponding Rep protein expression elements, including a Rep protein coding sequence and a corresponding promoter.

[0089] The vector may further comprise a first terminator and a second terminator, located upstream and downstream of the target DNA expression cassette in a), respectively, and configured such that transcription outside the target DNA expression cassette in a) in the vector terminates upstream and downstream of the target DNA expression cassette in a); or located upstream and downstream of the target DNA sequence group in b), respectively, and configured such that transcription outside the target DNA sequence group in b) in the vector terminates upstream and downstream of the target DNA sequence group in b). The first terminator may be a forward terminator, a bidirectional terminator, or a reverse terminator. The second terminator may be a forward terminator, a bidirectional terminator, or a reverse terminator. In some embodiments, the first terminator and the second terminator may both be bidirectional terminators. The first terminator and the second terminator may be independently selected from the group consisting of rrnB T1 terminator, rrnB T2 terminator, lambda t0 terminator, TonB terminator, Amp terminator, T7 terminator, and CYC1 terminator. In some embodiments, the first terminator may be rrnB T1 terminator and rrnB T2 terminator. In some embodiments, the second terminator may be lambda t0 terminator.

[0090] The vector can also include a screening marker. The screening marker can be an antibiotic resistance gene, such as an ampicillin resistance gene, a tetracycline resistance gene, a chloramphenicol resistance gene, a kanamycin resistance gene or a streptomycin resistance gene. In particular, the screening marker is not present in the genome of the host cell to be transformed / transfected by the vector. In some embodiments, the screening marker is a kanamycin resistance gene. The vector can also include a promoter for the expression of the screening marker upstream of the screening marker.

[0091] The first ITR and the second ITR may be wild-type AAV-ITR, comprising, for example, the nucleotide sequence shown in SEQ ID NO:9.

[0092] The first ITR-derived sequence and the second ITR-derived sequence can form a palindromic structure similar to a natural AAV-ITR, for example, comprising a palindromic arm and a palindromic stem, or comprising only a palindromic stem, and retaining Rep protein binding ability. In some embodiments, the first ITR-derived sequence and the second ITR-derived sequence can comprise the nucleotide sequence shown in SEQ ID NO: 10, 11, or 12.

[0093] The first ITR or a sequence derived therefrom and the second ITR or a sequence derived therefrom may comprise the same or different nucleotide sequences.

[0094] The vector may be a circular vector, a non-viral vector, or a plasmid vector.

[0095] The vector does not contain the lac promoter capable of initiating transcription.

[0096] The target DNA expression cassette of a) can include an IIS type restriction endonuclease (IIS enzyme) recognition site. The target DNA sequence group of b) can include an IIS enzyme recognition site. Except for the target DNA expression cassette of a) or the target DNA sequence group of b), the vector does not include the same IIS enzyme recognition site as a) the target DNA expression cassette and b) the target DNA sequence group. The target DNA sequence can be any DNA sequence to be expressed, such as an mRNA coding sequence and a poly (A) tail coding sequence. The target DNA sequence can include a DNA sequence that can form a non-B-type DNA configuration. The target DNA sequence can include a cloning site, such as a multiple cloning site.

[0097] The target DNA expression cassette in a) can be an expression cassette suitable for expressing the target DNA sequence in eukaryotic cells and can include components that enable expression of the target DNA sequence in eukaryotic cells. The promoter included in the target DNA expression cassette in a) can be a promoter that enables expression of the target DNA sequence in eukaryotic cells. The target DNA expression cassette in a) can also include enhancers, transcription terminators, etc., particularly enhancers and transcription terminators that enable expression of the target DNA sequence in eukaryotic cells. Transcription terminators suitable for expression of the target DNA sequence in eukaryotic cells can be selected from SV40 poly(A), hGH poly(A), BGH poly(A), and rbGlob poly(A). In some embodiments, the target DNA expression cassette in a) can be an expression cassette suitable for expressing mRNA in eukaryotic cells and can include optional enhancers, promoters, 5' UTRs, open reading frames (ORFs), 3' UTRs, poly(A) tail coding sequences, and transcription terminators. The target DNA expression cassette in a) can also include restriction sites for vector linearization.

[0098] In some embodiments, the vector may comprise:

[0099] i) the origin of replication,

[0100] ii) a) a target DNA expression cassette comprising, in order, a first ITR or a sequence derived therefrom, a promoter, a target DNA sequence, and a second ITR or a sequence derived therefrom, or b) a target DNA sequence group comprising, in order, a first ITR or a sequence derived therefrom, a target DNA sequence, and a second ITR or a sequence derived therefrom, and

[0101] iii) a selection marker and a promoter for controlling the expression of the selection marker,

[0102] The replication origin may be spaced at least 500 bp or 700 bp from a) the DNA expression cassette or b) the target DNA sequence group in the replication direction.

[0103] In some embodiments, the carrier backbone may comprise:

[0104] i) the origin of replication,

[0105] ii) a first terminator,

[0106] iii) a) a target DNA expression cassette comprising, in sequence, a first ITR or a sequence derived therefrom, a promoter, a target DNA sequence, and a second ITR or a sequence derived therefrom, or b) a target DNA sequence group comprising, in sequence, a first ITR or a sequence derived therefrom, a target DNA sequence, and a second ITR or a sequence derived therefrom,

[0107] iv) a second terminator, and

[0108] v) a selection marker and a promoter for controlling the expression of the selection marker,

[0109] wherein the first terminator and the second terminator are respectively located upstream and downstream of the target DNA expression cassette in a), and are configured such that transcription of the target DNA expression cassette other than the target DNA expression cassette in a) in the vector is terminated upstream and downstream of the target DNA expression cassette in a), or the first terminator and the second terminator are respectively located upstream and downstream of the target DNA sequence group in b), and are configured such that transcription of the target DNA sequence group other than the target DNA sequence group in b) in the vector is terminated upstream and downstream of the target DNA sequence group in b).

[0110] The replication origin may be spaced at least 500 bp or 700 bp from a) the DNA expression cassette or b) the target DNA sequence group in the replication direction.

[0111] In some embodiments, the vector may contain, in order, a pUC replication origin, an rrnB T1 terminator and an rrnB T2 terminator, a first ITR or a sequence derived therefrom, a CMV enhancer, a chicken β-actin promoter, a DNA sequence of interest, a bGH poly (A) signal, a second ITR or a sequence derived therefrom, a lambda t0 terminator, and a kanamycin resistance element.

[0112] Accordingly, the present application provides a carrier skeleton, which may include:

[0113] i) origin of replication, and

[0114] ii) a) a DNA sequence group comprising, in order, a first ITR or a sequence derived therefrom, a cloning site, and a second ITR or a sequence derived therefrom, or b) a DNA expression cassette comprising, in order, a first ITR or a sequence derived therefrom, a promoter, a cloning site, and a second ITR or a sequence derived therefrom.

[0115] The origin of replication can be spaced at least 150bp, 200bp, 300bp, 500bp, 700bp, 800bp, 900bp, 1000bp, 1100bp, 1200bp, 1300bp, 1400bp, 1500bp, 1600bp, or 1700bp in the direction of replication from the DNA sequence set of a) or the DNA expression cassette of b). In some embodiments, the origin of replication can be spaced at least 500bp in the direction of replication from the DNA sequence set of a) or the DNA expression cassette of b). In some embodiments, the origin of replication can be spaced at least 700bp in the direction of replication from the DNA sequence set of a) or the DNA expression cassette of b).

[0116] The origin of replication can enable the vector to replicate in a suitable host cell. Suitable host cells can be prokaryotes such as Escherichia coli, or eukaryotic cells. The origin of replication can be selected from the group consisting of the pMB1 origin of replication, the pBR322 origin of replication, the ColE1 origin of replication, the R6K origin of replication, the p15A origin of replication, the pSC101 origin of replication, the pUC origin of replication, the pET origin of replication, and derivatives of these origins of replication. These derivatives of the origin of replication can also enable the vector to replicate in a suitable host cell. In some embodiments, the origin of replication can be the pUC origin of replication.

[0117] The vector backbone can also comprise a second origin of replication, which can be spaced at least 150bp, 200bp, 300bp, 500bp, 700bp, 800bp, 900bp, 1000bp, 1100bp, 1200bp, 1300bp, 1400bp, 1500bp, 1600bp, or 1700bp apart in the replication direction from the DNA sequence set of a) or the DNA expression cassette of b). This second origin of replication can allow the vector to replicate in a suitable host cell, which can be a prokaryotic cell such as Escherichia coli or a eukaryotic cell.

[0118] Depending on the type of the replication origin, the vector may also contain corresponding Rep protein expression elements, including a Rep protein coding sequence and a corresponding promoter.

[0119] The carrier skeleton can also comprise the first terminator and the second terminator, be positioned respectively at the upstream and downstream of the DNA sequence group of a), and be arranged so that the transcription outside the DNA sequence group of a) in the carrier terminates at the upstream and downstream of the DNA sequence group of a), or the first terminator and the second terminator are positioned respectively at the upstream and downstream of the DNA expression cassette of b), and be arranged so that the transcription outside the DNA expression cassette of b) in the carrier terminates at the upstream and downstream of the DNA expression cassette of b).The first terminator can be a forward terminator, a bidirectional terminator or a reverse terminator.The second terminator can be a forward terminator, a bidirectional terminator or a reverse terminator.In some embodiments, the first terminator and the second terminator can both be bidirectional terminators.The first terminator and the second terminator can independently be selected from rrnB T1 terminator, rrnB T2 terminator, lambda t0 terminator, TonB terminator, Amp terminator, T7 terminator and CYC1 terminator.In some embodiments, the first terminator can be rrnB T1 terminator and rrnB T2 terminator. In some embodiments, the second terminator can be a lambda t0 terminator.

[0120] The cloning site in the DNA sequence group in a) of the vector backbone can be a multiple cloning site. The cloning site in the DNA expression cassette in b) of the vector backbone can be a multiple cloning site.

[0121] In the DNA sequence group of a), any sequence can be inserted into the cloning site, in particular, a target DNA sequence, or a promoter and a target DNA sequence. The target DNA sequence can be any DNA sequence to be expressed, such as an mRNA coding sequence.

[0122] In the DNA expression cassette of b), any sequence, in particular a target DNA sequence, can be inserted into the cloning site. The target DNA sequence can be any DNA sequence to be expressed, such as an mRNA coding sequence.

[0123] The vector backbone can also include a screening marker. The screening marker can be an antibiotic resistance gene, such as an ampicillin resistance gene, a tetracycline resistance gene, a chloramphenicol resistance gene, a kanamycin resistance gene or a streptomycin resistance gene. In particular, the screening marker is not present in the genome of the host cell to be transformed / transfected by the vector. In some embodiments, the screening marker is a kanamycin resistance gene. The vector can also include a promoter for the expression of the screening marker upstream of the screening marker.

[0124] The first ITR and the second ITR may be wild-type AAV-ITR, comprising, for example, the nucleotide sequence shown in SEQ ID NO:9.

[0125] The first ITR-derived sequence and the second ITR-derived sequence can form a palindromic structure similar to a natural AAV-ITR, for example, comprising a palindromic arm and a palindromic stem, or comprising only a palindromic stem, and retaining Rep protein binding ability. In some embodiments, the first ITR-derived sequence and the second ITR-derived sequence can comprise the nucleotide sequence shown in SEQ ID NO: 10, 11, or 12.

[0126] The first ITR or a sequence derived therefrom and the second ITR or a sequence derived therefrom may comprise the same or different nucleotide sequences.

[0127] The vector backbone may be a circular vector backbone, a non-viral vector backbone, or a plasmid vector backbone.

[0128] The vector backbone does not contain a lac promoter capable of initiating transcription.

[0129] The DNA expression cassette in b) can be an expression cassette suitable for expressing the DNA sequence in eukaryotic cells and can include components that enable expression of the DNA sequence in eukaryotic cells. The promoter included in the DNA expression cassette in b) can be a promoter that enables expression of the DNA sequence in eukaryotic cells. The target DNA expression cassette in b) can also include enhancers, transcription terminators, etc., particularly enhancers and transcription terminators that enable expression of the DNA sequence in eukaryotic cells. Transcription terminators suitable for expression of the DNA sequence in eukaryotic cells can be selected from SV40 poly(A), hGH poly(A), BGH poly(A), and rbGlob poly(A). In some embodiments, the target DNA expression cassette in b) can be an expression cassette suitable for expressing mRNA in eukaryotic cells and can include optional enhancers, promoters, 5' UTRs, open reading frames (ORFs), 3' UTRs, and transcription terminators. The DNA expression cassette in b) can also include restriction sites for vector linearization.

[0130] In some embodiments, the carrier backbone may comprise:

[0131] i) the origin of replication,

[0132] ii) a) a DNA sequence group comprising, in order, a first ITR or a sequence derived therefrom, a cloning site, and a second ITR or a sequence derived therefrom, or b) a DNA expression cassette comprising, in order, b) a first ITR or a sequence derived therefrom, a promoter, a cloning site, and a second ITR or a sequence derived therefrom, and

[0133] iii) a selection marker and a promoter for controlling the expression of the selection marker,

[0134] The replication origin may be separated from a) the DNA sequence group, or b) the DNA expression cassette by at least 500 bp, or 700 bp in the replication direction.

[0135] In some embodiments, the carrier backbone may comprise:

[0136] i) the origin of replication,

[0137] ii) a first terminator,

[0138] iii) a) a DNA sequence group comprising, in order, a) a first ITR or a sequence derived therefrom, a cloning site, and a second ITR or a sequence derived therefrom, or b) a DNA expression cassette comprising, in order, b) a first ITR or a sequence derived therefrom, a promoter, a cloning site, and a second ITR or a sequence derived therefrom,

[0139] iv) a second terminator, and

[0140] v) a selection marker and a promoter for controlling the expression of the selection marker,

[0141] wherein the first terminator and the second terminator are located upstream and downstream of the DNA sequence group a), respectively, and are configured so that transcription from other than the DNA sequence group a) in the vector is terminated upstream and downstream of the DNA sequence group a), or

[0142] The first terminator and the second terminator are located upstream and downstream of the DNA expression cassette of b), respectively, and are configured so that transcription from outside the DNA expression cassette of b) in the vector is terminated upstream and downstream of the DNA expression cassette of b).

[0143] The replication origin may be separated from a) the DNA sequence group, or b) the DNA expression cassette by at least 500 bp, or 700 bp in the replication direction.

[0144] In some embodiments, the vector backbone can comprise, in order, a pUC replication origin, an rrnB T1 terminator and an rrnB T2 terminator, a first ITR or a sequence derived therefrom, a CMV enhancer, a chicken β-actin promoter, a cloning site, a bGH poly (A) signal, a second ITR or a sequence derived therefrom, a lambda t0 terminator, and a kanamycin resistance element.

[0145] In a third aspect, the present application provides a method for preparing the vector of the present application, comprising synthesizing the vector from scratch according to the design of the vector skeleton of the present application.

[0146] The present application provides a method for preparing the vector skeleton of the present application, comprising synthesizing the vector from scratch according to the design of the vector skeleton of the present application.

[0147] The present application also provides a method for modifying a vector or a vector skeleton, comprising i) providing a vector or a vector skeleton, and ii) modifying the vector or the vector skeleton so that the modified vector or the vector skeleton comprises:

[0148] a) Copy starting point,

[0149] b) a first terminator,

[0150] c) ci) cloning site,

[0151] c-ii) a DNA expression cassette comprising a promoter and a cloning site,

[0152] c-iii) a DNA expression cassette comprising a promoter, a cloning site and a DNA sequence capable of forming a non-B-form DNA conformation,

[0153] c-iv) a target DNA sequence comprising a DNA sequence capable of forming a non-B-form DNA conformation,

[0154] cv) a target DNA expression cassette comprising a promoter and a target DNA sequence, wherein the target DNA sequence comprises a DNA sequence capable of forming a non-B-form DNA conformation,

[0155] c-vi) a DNA sequence group comprising, in order, the first ITR or its derivative sequence, a cloning site,

[0156] and a second ITR or a derivative thereof,

[0157] c-vii) a DNA expression cassette comprising, in order, a first ITR or a sequence derived therefrom, a promoter, a cloning site, and a second ITR or a sequence derived therefrom,

[0158] c-viii) a target DNA expression cassette comprising, in order, a first ITR or a sequence derived therefrom, a promoter, a target DNA sequence, and a second ITR or a sequence derived therefrom, or

[0159] c-ix) a target DNA sequence group comprising, in order, a first ITR or a derivative sequence thereof, a target DNA sequence, and a second ITR or a derivative sequence thereof,

[0160] d) a second terminator, and

[0161] e) a selection marker and a promoter for controlling the expression of the selection marker,

[0162] wherein the first terminator and the second terminator are located upstream and downstream of the sequence of c), respectively, and are configured such that transcription outside the sequence of c) terminates upstream and downstream of the sequence of c),

[0163] Optionally, the replication origin is separated from the cloning site of ci), the cloning site in the DNA expression cassette of c-ii), the DNA sequence capable of forming a non-B-form DNA configuration in the DNA expression cassette of c-iii), the DNA sequence capable of forming a non-B-form DNA configuration in the target DNA sequence of c-iv), the DNA sequence capable of forming a non-B-form DNA configuration in the target DNA expression cassette of cv), the DNA sequence group of c-vi), the DNA expression cassette of c-vii), the target DNA expression cassette of c-viii), or the target DNA sequence group of c-ix) by at least 150 bp, 200 bp, 300 bp, 500 bp or 700 bp in the replication direction, and

[0164] Optionally, the vector does not contain an active lac promoter.

[0165] The cloning site in the sequence of c) may be a multiple cloning site.

[0166] Specifically, the present application also provides a method for modifying a vector or a vector skeleton, comprising:

[0167] i) providing a vector or vector backbone comprising a) an origin of replication, and b) bi) a cloning site, b-ii) a DNA expression cassette comprising a promoter and a cloning site, b-iii) a DNA expression cassette comprising a promoter, a cloning site, and a DNA sequence capable of forming a non-B-form DNA configuration, b-iv) a target DNA sequence comprising a DNA sequence capable of forming a non-B-form DNA configuration, bv) a target DNA expression cassette comprising a promoter and a target DNA sequence, the target DNA sequence comprising a DNA sequence capable of forming a non-B-form DNA configuration, b-vi) a DNA sequence a) a sequence group comprising, in sequence, a first ITR or a sequence derived therefrom, a cloning site, and a second ITR or a sequence derived therefrom, b-vii) a DNA expression cassette comprising, in sequence, a first ITR or a sequence derived therefrom, a promoter, a cloning site, and a second ITR or a sequence derived therefrom, b-viii) a target DNA expression cassette comprising, in sequence, a first ITR or a sequence derived therefrom, a promoter, a target DNA sequence, and a second ITR or a sequence derived therefrom, or b-ix) a target DNA sequence group comprising, in sequence, a first ITR or a sequence derived therefrom, a target DNA sequence, and a second ITR or a sequence derived therefrom; and

[0168] ii) adding a first terminator and a second terminator to the vector or vector backbone so that they are located upstream and downstream of the sequence b), respectively, and configured to terminate transcription outside the sequence b) upstream and downstream of the sequence b).

[0169] The method may further include spacing the origin of replication by at least 150 bp, 200 bp, 300 bp, 500 bp, or 700 bp in the direction of replication from the cloning site of bi), the cloning site in the DNA expression cassette of b-ii), the DNA sequence capable of forming a non-B-form DNA configuration in the DNA expression cassette of b-iii), the DNA sequence capable of forming a non-B-form DNA configuration in the target DNA sequence of b-iv), the DNA sequence capable of forming a non-B-form DNA configuration in the target DNA expression cassette of bv), the DNA sequence group of b-vi), the DNA expression cassette of b-vii, the target DNA expression cassette of b-viii, or the target DNA sequence group of b-ix.

[0170] Where the vector or vector backbone further comprises a lac promoter, the method further comprises inactivating the lac promoter.

[0171] The DNA sequence capable of forming a non-B-form DNA configuration can be a forward repeat sequence, an inverted repeat sequence, or a single purine-single pyrimidine sequence, including but not limited to polyadenine (poly(A)), polyguanine (poly(G)), polycytosine (poly(C)), or polythymine (poly(T)), shRNA coding sequence, and ITR sequence. The forward repeat sequence can be a poly(A) tail coding sequence. The single purine-single pyrimidine sequence can be a poly(A) tail coding sequence. The inverted repeat sequence can be an ITR or a sequence derived therefrom. In some embodiments, the DNA sequence capable of forming a non-B-form DNA configuration can be a poly(A) coding sequence, an ITR or a sequence derived therefrom.

[0172] The origin of replication can enable the vector or vector backbone to replicate in a suitable host cell. Suitable host cells can be prokaryotes such as Escherichia coli, or eukaryotic cells. The origin of replication can be selected from the group consisting of the pMB1 origin of replication, the pBR322 origin of replication, the ColE1 origin of replication, the R6K origin of replication, the p15A origin of replication, the pSC101 origin of replication, the pUC origin of replication, the pET origin of replication, and derivatives of these origins of replication. These derivatives of the origin of replication can also enable the vector to replicate in a suitable host cell. In some embodiments, the origin of replication can be the pUC origin of replication.

[0173] Depending on the type of the replication origin, the vector may also contain corresponding Rep protein expression elements, including a Rep protein coding sequence and a corresponding promoter.

[0174] In some embodiments, the first terminator can be a forward terminator, a bidirectional terminator, or a reverse terminator. The second terminator can be a forward terminator, a bidirectional terminator, or a reverse terminator. In some embodiments, the first terminator and the second terminator can both be bidirectional terminators. The first terminator and the second terminator can be independently selected from rrnB T1 terminator, rrnB T2 terminator, lambda t0 terminator, TonB terminator, Amp terminator, T7 terminator, and CYC1 terminator. In some embodiments, the first terminator can be rrnB T1 terminator and rrnB T2 terminator. In some embodiments, the second terminator can be lambda t0 terminator.

[0175] The vector or vector backbone can comprise a screening marker. The screening marker can be an antibiotic resistance gene, such as an ampicillin resistance gene, a tetracycline resistance gene, a chloramphenicol resistance gene, a kanamycin resistance gene or a streptomycin resistance gene. In particular, the screening marker is not present in the genome of the host cell to be transformed / transfected by the vector. In some embodiments, the screening marker is a kanamycin resistance gene. The vector can also comprise a promoter for the expression of the screening marker upstream of the screening marker.

[0176] The vector or vector backbone may be circular. The vector or vector backbone may be of non-viral origin. The vector or vector backbone may be a plasmid vector or a plasmid vector backbone.

[0177] In a fourth aspect, the present application provides a composition comprising the carrier skeleton or carrier of the present application, including the carrier skeleton or carrier prepared by the method of the present application, and the carrier skeleton or carrier modified by the method of the present application.

[0178] In the fifth aspect, the present application provides the vector backbone or vector of the present application, including those prepared by the method of the present application and those modified by the method of the present application, and their use in replicating and / or expressing target DNA sequences, especially DNA sequences containing DNA sequences capable of forming non-B-type DNA configurations (such as ITRs, or poly (A) tail coding sequences).

[0179] Specifically, the present application provides a method for replicating a target DNA sequence, comprising:

[0180] i) providing the vector described in the first aspect of the present application,

[0181] Providing the vector backbone described in the first aspect of the present application, inserting a target DNA sequence, or a promoter and a target DNA sequence, into the cloning site of a), inserting a target DNA sequence into the cloning site of b), inserting a target DNA sequence into the cloning site of the DNA expression cassette of c), or inserting a target DNA sequence into the cloning site of the DNA expression cassette of d), to obtain a new vector,

[0182] Providing the vector described in the second aspect of the present application, or

[0183] Providing the vector backbone described in the second aspect of the present application, inserting a target DNA sequence, or a promoter and a target DNA sequence, into the cloning site of the DNA sequence group in a), or inserting a target DNA sequence into the cloning site of b) the DNA expression cassette to obtain a new vector;

[0184] ii) transforming / transfecting the vector of step i) into a host cell;

[0185] iii) culturing the host cells under conditions conducive to vector replication.

[0186] The present application also provides a method for expressing a target DNA sequence, comprising:

[0187] i) providing a vector comprising a) a target DNA expression cassette as described in the first aspect of the present application, or

[0188] Providing the vector backbone described in the first aspect of the present application, inserting a promoter and a target DNA sequence at the cloning site of a), inserting a target DNA sequence at the cloning site of b), inserting a target DNA sequence at the cloning site of the DNA expression cassette of c), or inserting a target DNA sequence at the cloning site of the DNA expression cassette of d), to obtain a new vector; and

[0189] ii) transforming / transfecting the vector of step i) into a host cell, culturing the host cell under conditions conducive to the expression of the target DNA sequence, or linearizing the vector of step i) and performing in vitro transcription; or

[0190] i) providing the vector comprising a) the target DNA expression cassette as described in the second aspect of the present application,

[0191] or providing the vector backbone described in the second aspect of the present application, inserting a promoter and a target DNA sequence at the cloning site of the DNA sequence group in a), or inserting a target DNA sequence at the cloning site of b) the DNA expression cassette to obtain a new vector,

[0192] ii) transforming / transfecting the vector of step i) into a host cell, and culturing the host cell under conditions conducive to the expression of the target DNA sequence,

[0193] Linearizing the vector of step i) and performing in vitro transcription; or

[0194] The vector of step i) is co-transformed / transfected with a packaging plasmid and a helper plasmid into a host cell, the host cell is cultured under conditions conducive to vector packaging, the packaged vector is recovered, the host cell is transformed / transfected, and the host cell is cultured under conditions conducive to expression of the target DNA sequence.

[0195] The method may further comprise, between step i) and step ii), transforming / transfecting the vector of step i) into a host cell, and culturing the host cell under conditions conducive to vector replication.

[0196] In a sixth aspect, the present application provides the use of the vector backbone or vector of the present application, including those prepared by the method of the present application and those modified by the method of the present application, in preparing a recombinant adeno-associated virus vector (rAAV).

[0197] Specifically, the present application provides a method for preparing a recombinant adeno-associated virus vector, comprising:

[0198] i) providing the vector described in the second aspect of the present application, or

[0199] Providing the vector backbone described in the second aspect of the present application, inserting a target DNA sequence, or a promoter and a target DNA sequence, into the cloning site of the DNA expression cassette in a), or inserting a target DNA sequence into the cloning site of the DNA expression cassette in b), to obtain a new vector;

[0200] ii) optionally, transforming / transfecting the vector of step i) into a host cell, and culturing the host cell under conditions conducive to vector replication; and

[0201] iii) harvesting the vector from step i) or ii), co-transforming / transfecting host cells with the vector, a packaging plasmid, and a helper plasmid, and culturing the host cells under conditions conducive to vector packaging.

[0202] Based on the following specific description and examples, other features and advantages of the current disclosure will become clearer, and specific description and examples should not be interpreted as restrictive. The contents of all documents, Genbank records, patents and published patent applications cited in this application are expressly included in this article by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0203] The following detailed description is given by way of example but is not intended to limit the present invention to the specific embodiments described, and can be better understood in conjunction with the accompanying drawings.

[0204] FIG1 shows a schematic diagram of the pUC57-kana test plasmid.

[0205] Figure 2 shows the sequencing schematics of a failed clone (A) lacking the poly(A) tail coding sequence, a failed clone (B) with an impure poly(A) tail coding sequence and a sequencing bottom peak that is 20% higher than the main peak, and a positive clone (C) with a relatively pure poly(A) tail coding sequence and a sequencing bottom peak that is 20% lower than the main peak.

[0206] FIG3 shows the single cloning success rate of pUC57-kana plasmids with different lengths of spacer sequences between the Ori and poly(A) tail coding sequences.

[0207] Figure 4 shows a schematic diagram of the pUC57-kana plasmid containing a terminator in the 247 bp spacer sequence between the Ori and poly(A) tail coding sequences.

[0208] FIG5 shows a schematic diagram of sequencing of the pUC57-kana plasmid without a terminator (A) or with a terminator (B) in the 247 bp spacer sequence, and the single cloning success rate of the two plasmids (C).

[0209] FIG6 shows a schematic diagram of the pUC57-kana plasmid in which the lacP functional region is disrupted by a 465 bp spacer sequence.

[0210] FIG7 shows the effect of the lacP functional region on the success rate of constructing a single clone of a plasmid containing a poly(A) tail coding sequence.

[0211] FIG8 shows a map of the GS-MK plasmid.

[0212] FIG9 shows a map of the GS-PV plasmid.

[0213] FIG10 shows the sequencing peak profiles of GS-MK vectors inserted with different poly(A) tail encoding sequences.

[0214] FIG11 shows the sequencing peak profiles of GS-PV vectors inserted with different poly(A) tail encoding sequences.

[0215] FIG12 shows the sequencing peak profiles of GS-CMV vectors inserted with different poly(A) tail encoding sequences.

[0216] FIG. 13 shows the success rates of single cloning of pUC57-kana, GS-MK, PVAX1, GS-PV, and GS-CMV containing poly(A) tail coding sequences.

[0217] FIG. 14 shows the passage stability of pUC57-kana, GS-MK, PVAX1, GS-PV, and GS-CMV containing a poly(A) tail coding sequence.

[0218] FIG. 15 shows the yields of pUC57-kana, GS-MK, PVAX1, GS-PV, and GS-CMV containing poly(A) tail coding sequences.

[0219] FIG16 shows the purity of in vitro transcription products of pUC57-kana, GS-MK, PVAX1, GS-PV, and GS-CMV containing poly(A) tail coding sequences.

[0220] FIG17 shows the protein expression levels of mRNAs transcribed from pUC57-kana, GS-MK, PVAX1, GS-PV, and GS-CMV containing poly(A) tail coding sequences.

[0221] Figure 18 shows the maps of four lacP-containing plasmids.

[0222] Figure 19 shows a schematic diagram of the pUC57-kana vector with added ITR sequence (A), and a schematic diagram of the vector with added ITR sequence, adjusted Ori position, destroyed lacP functional region, and added terminator sequence downstream of the 5'ITR position and upstream of the 3'ITR (B).

[0223] FIG20 shows a schematic diagram of the pAAV-tTA plasmid (A), and a schematic diagram of a vector in which the promoter is replaced and terminator sequences are added downstream of the 5' ITR and upstream of the 3' ITR (B).

[0224] Figure 21 shows the restriction enzyme electrophoresis patterns of pUC57 kana, GenS-pUC57-AAV-NoTer, and GenS-pUC57-AAV-Ter after gene cloning. Lanes 1-3: pUC57 kana vector, SmaI digestion product, and a standard-sized DNA marker; Lanes 4-6: GenS-pUC57-AAV-NoTer vector, SmaI digestion product, and a standard-sized DNA marker; Lanes 7-9: GenS-pUC57-AAV-Ter vector, SmaI digestion product, and a standard-sized DNA marker. DETAILED DESCRIPTION

[0225] Unless otherwise specified, the terms used herein have the ordinary meanings in dictionaries, textbooks, technical reference books, or as generally understood by those skilled in the art. The following descriptions of certain terms are intended only to facilitate understanding of this application and are not intended to be limiting of these terms unless otherwise specified.

[0226] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "a vector" may include a plurality of such vectors.

[0227] The term "cloning vector" refers to a self-replicating DNA molecule containing genetic elements such as an origin of replication, a selection marker, and a cloning site. It can carry DNA fragments into host cells and amplify these fragments. A cloning vector may contain a nucleic acid sequence, such as an origin of replication, that enables the vector to replicate autonomously in one or more host cells.

[0228] The term "expression vector" refers to a vector obtained by adding elements related to expression regulation, such as a promoter, a ribosome binding site, a terminator, etc., to a cloning vector.

[0229] A vector backbone is a vector that contains essential replication and expression elements, as well as other necessary elements. A DNA sequence of interest can be inserted into the vector backbone via cloning sites or replaced by homologous recombination, thereby enabling replication and expression of the DNA sequence of interest.

[0230] "Origin of replication" or "Ori" refers to the site where DNA replication starts, which controls the replication of the plasmid and determines the host properties and copy number of the plasmid. Ori is usually rich in adenine (A) and thymine (T), easily melts, and provides space for replication regulatory elements such as Rep protein to bind. Common replication origins include pUC (nucleotide sequence shown in SEQ ID NO: 23), ColE1, pMB1, pSC101, R6K, etc. Depending on the control of the replication origin, vectors can be divided into relaxed and tight types. In tight plasmid vectors, the replication of the plasmid and the replication of the host cell chromosome appear to be controlled by the same factors. The host cell replicates once and the plasmid also replicates once. There are only 1-2 plasmids in each host cell. In relaxed plasmids, when the host cell chromosome replication stops, it can still continue to replicate. It appears to be controlled by factors different from chromosome replication. Each cell generally contains dozens to hundreds of plasmids. In addition, depending on the design of the replication origin regulatory elements in the vector, the vector can be narrow host type or wide host type. In narrow-host vectors, Ori relies on replication elements or Rep proteins provided by the host cell, while in broad-host vectors, the vector autonomously contains Rep protein expression elements. "Rep proteins" can bind to replication origins, move in a specific direction, and separate the double-stranded DNA. Vectors contain one or more replication origins for autonomous replication in one or more host cells.

[0231] "Screening marker" refers to a gene in a vector that acts as a specific marker and can be used to verify whether the vector has been successfully transformed / transfected into a host cell. The screening marker can be inherent to the vector or recombinantly added. The screening marker can be a resistance gene, preferably a resistance gene that the host cell does not possess. In this way, when the vector is successfully introduced into a host cell, the host cell can be given corresponding resistance, thereby determining that the vector has entered the host cell. Commonly used screening markers include ampicillin (Ampr) resistance gene, chloramphenicol (Camr) resistance gene, kanamycin (Kanr) resistance gene, tetracycline (Tetr) resistance gene, puromycin (Puro) resistance gene, G418 resistance gene, hygromycin beta (Hygr) resistance gene, bleomycin resistance gene, blasticidin (specifically inhibiting protein synthesis in prokaryotes and eukaryotes by interfering with the formation of peptide bonds in ribosomes) resistance gene, etc.

[0232] "Cloning site" refers to a DNA fragment contained in a vector, which contains one or more restriction endonuclease recognition sites. After being treated with the corresponding restriction endonuclease, the cloning site can allow the insertion of exogenous DNA treated with the same restriction endonuclease. According to the number of restriction endonuclease recognition sites on the sequence, the cloning site can be divided into a single cloning site and a multiple cloning site (MCS). The cloning site can include recognition sites for NheI, BmtI, AflII, HindIII, Acc65I, KpnI, BamHI, ApoI, RcoRI, PstI, EcoRV, BstXI and / or NotI. Those skilled in the art can routinely determine the sequence of the cloning site. The cloning site can include recognition sites for IIS type restriction endonucleases. IIS type restriction endonucleases are a group of relatively special endonucleases that cut DNA at a specific position downstream of the recognition site, and there are no requirements for the sequence of the cleavage site. In some embodiments, the target DNA sequence to be inserted or inserted into the vector backbone, such as the end of the mRNA coding sequence and the poly(A) tail coding sequence, contains an IIS enzyme recognition site, while the vector backbone does not contain a corresponding IIS enzyme recognition site. For example, IIS sites such as BsaI, BspQI, BspMI, BpiI, and BsmbI can be removed through synonymous mutation. In this way, after linearization of the vector containing the target DNA sequence, the IIS restriction endonuclease recognizes only the region outside the poly(A) tail coding sequence and cuts within the poly(A) coding sequence. First, the resulting enzyme-cleaved fragments will not appear messy due to the presence of multiple IIS enzyme cleavage sites. Second, the polyT sequence in the enzyme-cleaved fragments will not be added to non-T sequences, and the corresponding poly(A) tails obtained will also be pure. Pure poly(A) tails can maintain high mRNA protein expression levels (Holtkamp S et al., (2006) Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells. Blood. 108(13): 4009-4017). This design also facilitates Golden Gate cloning of the vector.

[0233] "Promoter" refers to a DNA sequence that RNA polymerase recognizes, binds to, and initiates transcription. It contains conserved sequences required for RNA polymerase specific binding and transcription initiation. Most promoters are located upstream of the gene transcription start point and are not transcribed themselves. Promoters can be divided into constitutive promoters, tissue / cell-specific promoters, and inducible promoters. Constitutive promoters are also called non-specific expression promoters. Under their regulation, gene expression does not differ significantly in different cells, tissues, organs, or developmental stages. Common promoters of this type include Ubc, CMV, SV40, EF1a, U6, CAG (composed of the CMV enhancer and the chicken β-actin promoter), etc. Under the regulation of tissue / cell-specific promoters, exogenous genes are specifically expressed only in specific cells. An inducible expression promoter refers to a promoter that begins to significantly increase the transcription level of a gene under the stimulation of certain specific physical or chemical signals. The most common one is the tetracycline-inducible expression promoter.

[0234] An "enhancer" is a region on DNA that can bind to transcription factors. Once bound, these regions enhance gene transcription. Enhancers can be located upstream or downstream of a gene and are not necessarily located close to the gene they are intended to affect. Common enhancers include the CMV enhancer.

[0235] A "terminator" is a DNA sequence that terminates RNA polymerase transcription and initiates the separation of newly synthesized RNA from the transcription machinery. Terminators are typically located after the 3' regulatory sequence of a gene. Prokaryotic terminators can be divided into two categories: those that rely on protein cofactors for termination, commonly known as p-factors (Rho factors), and those that are independent of protein cofactors. These terminators share some common sequence characteristics: a GC-rich inverted repeat sequence that causes the transcribed RNA to form a hairpin structure, disrupting the normal structure of the 5' end of the RNA-DNA hybrid and pausing RNA polymerase transcription. The GC inverted repeat sequence is followed by a sequence consisting of 4-8 A residues, resulting in oligonucleotides in the transcript. This creates an unstable U·A region at the 3' end of the RNA-DNA hybrid, which, upon release, forms a weakly linked polyuracil ring. Almost all commonly used bacterial expression vectors use terminators that are independent of the rho factor, including naturally occurring terminators such as T7 and rrnB, and engineered, high-efficiency terminators such as T0. No terminator can initiate the termination step with 100% efficiency, but some engineered terminators can achieve termination efficiencies approaching 95%.

[0236] In this application, terminators suitable for expressing DNA sequences in eukaryotic cells may be referred to as "transcription terminators" in some cases. A eukaryotic terminator refers to a DNA fragment located at the end of a gene or operon that interrupts transcription in a eukaryotic system and binds to a tailing enzyme to promote polyadenylation. Commonly used eukaryotic terminators include SV40 poly(A), hGH poly(A), bGH poly(A), and rbGlob poly(A) signals. "bGH poly(A) signal" refers to the bovine growth factor (BGH) polyadenylation signal sequence, "hGH poly(A) signal" refers to the human growth factor (hGH) polyadenylation signal sequence, "SV40 poly(A) signal" refers to the simian vacuole virus polyadenylation signal sequence, and rbGlob poly(A) signal refers to the rabbit β-globin polyadenylation signal sequence, all of which can effectively terminate transcription and promote mRNA polyadenylation.

[0237] As used herein, a "DNA expression cassette" or "target DNA expression cassette" refers to an expression component comprising a promoter, a target DNA sequence (or a cloning site for insertion of the target DNA sequence), and an optional bGH poly(A) signal. When the DNA expression cassette is inserted into a vector containing the target DNA sequence, transcription of the target DNA sequence, and possibly further translation, can be performed under certain conditions. As used herein, a "DNA expression cassette" or "target DNA expression cassette," in some embodiments, refers to an expression cassette suitable for expressing a DNA sequence in eukaryotic cells.

[0238] The "target DNA sequence" in this application may refer to any DNA sequence fragment, in particular a DNA sequence fragment of interest. When the "target DNA sequence" is an mRNA coding sequence, it may include a 5'UTR, an open reading frame (ORF) and a 3'UTR. "Open reading frame" or "ORF" refers to a part of a gene sequence, which contains a nucleotide sequence that can encode a protein and is a theoretical amino acid coding region. The "target DNA sequence" can also be the coding sequence of a target protein, and the target protein can be any protein of interest, such as an antibody, enzyme, etc. The "target DNA sequence" in this application may include a DNA sequence that can form a non-B-type DNA configuration.

[0239] "In vitro transcription" or "IVT" refers to the process of producing RNA from a linear DNA template in a cell-free environment. The in vitro transcription reaction typically requires a purified linear DNA template containing a promoter, ribonucleotide triphosphates, a buffer system containing DTT and magnesium ions, and an appropriate amount of RNA polymerase.

[0240] "Host cell" encompasses cells that contain a vector and preferably support replication or replication and expression of the vector. The host cell can be a prokaryotic cell (e.g., E. coli) or a eukaryotic cell (e.g., yeast, insect, amphibian, or mammalian cell). In some embodiments, the host cell is an E. coli cell, such as an E. coli TOP10 cell, or the recombinant strain GenITRv1. In some embodiments, the host cell is a eukaryotic cell, such as a HEK293 cell.

[0241] The term "transformation" refers to the technique of introducing exogenous genes into prokaryotic host cells using a vector containing an exogenous gene (such as a recombinant plasmid), while "transfection" refers to the technique of introducing exogenous genes or vectors (such as recombinant plasmids) into eukaryotic cells (usually mammalian cells). Transformation and transfection are generally performed using competent cells, but can also be performed using methods such as artificial liposomes, gene guns, microinjection, and electroporation. "Competent" cells are cells that have been induced by physical and chemical methods to be in a state suitable for uptake and storage of foreign DNA. For example, cold treatment of Escherichia coli with divalent cations (such as CaCl2) can render the cell wall permeable to plasmid DNA.

[0242] "Adeno-associated virus" or "AAV" is a small, non-pathogenic virus of the Parvoviridae family that relies on a helper virus for replication. The AAV genome is approximately 5 kb and consists of a single-stranded DNA segment of either positive or negative polarity. The ends of the genome are short palindromic / inverted terminal repeats (ITRs), which are the only cis-acting elements in the genome. They can fold into a hairpin structure and serve as the starting point for viral DNA replication, participating in genome integration, rescue, replication, and packaging. As a DNA delivery vector, recombinant adeno-associated virus (rAAV) has become the most important vector for genetic research and gene therapy due to its high safety, low immunogenicity, wide range of applications, and long duration of action. To date, many different serotypes of AAV have been isolated from humans or primates. For example, the genome of AAV2 is 4680 nucleotides long and contains two open reading frames.

[0243] "Inverted terminal repeats" or "ITRs" refer to the inverted repeat sequences at the ends of the AAV genome that can form a palindromic structure. This is the only genetic component shared by recombinant AAV and wild-type AAV, and plays an important role in the replication, packaging, and sustained expression of exogenous genes in recombinant AAV. The ITR can be divided into seven segments in sequence: A, C, C', B, B', A', and D, where A and A' are reverse-complemented to form a large palindromic stem, B and B' are reverse-complemented to form a small palindromic arm, and C and C' are reverse-complemented to form a small palindromic arm. There is a Rep protein binding site in the A-A' region, another Rep protein binding site at the top of the B-B' region or the C-C' region, and a TRS sequence at the junction of A-A' and D. By reducing the B and B' palindromic arms, and / or the C and C' palindromic arms in the structure, while retaining the key one or two Rep protein binding sites and the TRS sequence, a simplified ITR can be obtained that retains the above-mentioned rAAV replication, packaging, and exogenous gene expression functions. The "ITR-derived sequence" in this application refers to an inverted palindromic sequence that can form a complete wild-type ITR structure or a partial wild-type ITR structure and retains the rAAV replication, packaging, and exogenous gene expression functions.

[0244] The lactose operon is a gene group involved in the decomposition of lactose, consisting of a regulatory gene (lacI), a promoter (lacP), an operator gene (lacO), and structural genes (lacZ, lacY, lacA). "lac promoter" or "lacP" in this application refers to the promoter in the lactose operon, which is the binding site for RNA polymerase during transcription initiation. There is a "leakage" in lacP, that is, the transcription of the promoter will not be completely "closed", and there is always some basic transcription (F. Jacob, J. Monod (1961) Genetic regulatory mechanisms in the synthesis of proteins, J. Mol. Biol. 3: 318-356). LacP is often constructed in plasmid cloning vectors for expressing recombinant proteins in bacteria and provides the ability of high-level expression for the introduction of genes. For expression cloning, constructs are usually constructed in medium- to high-copy number plasmids (such as pUC, pBluescript and pET vectors and their derivatives) to increase the copy number of the recombinant gene, which to some extent increases the "leaky" expression of lacP (Nielsen, Brent L., Van C. Willis, and Chin-Yo Lin. (2007) Western blot analysis to illustrate relative control levels of the lac and ara promoters in Escherichia coli, Biochemistry and Molecular Biology Education 35(2): 133-137).

[0245] "Non-B DNA configuration" refers to DNA configurations that are different from the typical right-hand helical double-strand (B-type), including A-type DNA configuration, Z-type DNA configuration, H-type DNA configuration, G-quadruplex, triplex, hairpin or cross, slip chain, etc. Studies have shown that these DNA configurations exist at least temporarily during DNA replication, transcription, repair or recombination, which will hinder the progress of these processes and increase the DNA mutation rate. The coding sequence of the poly (A) tail is composed of a large number of adenine (A) or thymine (T), which is easy to form H-type DNA configuration or triplex; inverted repeat sequences such as ITR or shRNA coding sequences are easy to form hairpin or cross structures; direct repeat sequences are easy to form slip chains (Guiblet WM et al., (2021) Non-B DNA: a major contributor to small-and large-scale variation in nucleotide substitution frequencies across the genome. Nucleic Acids Res. 49 (3): 1497-1516).

[0246] A "direct repeat" is a DNA sequence that repeats in the same direction, optionally with a spacer between the repeats. Direct repeats, particularly when rich in guanine (G), are prone to forming non-B-form DNA structures such as G-quadruplexes during replication and transcription.

[0247] "Inverted repeat sequence" refers to a sequence complementary in the opposite direction, with or without an intervening spacer. An example of an inverted repeat sequence is an ITR or shRNA coding sequence, which is prone to forming a hairpin or cruciform structure during replication and transcription.

[0248] A "purine-monopyrimidine sequence" refers to a double-stranded DNA sequence in which one strand is essentially or entirely purine, while the complementary strand is essentially or entirely pyrimidine. Specific examples include polyA sequences, polyT sequences, polyG sequences, and polyC sequences. These sequences can form H-shaped DNA structures or triplexes. Poly(A) tail encoding sequences, including but not limited to polyT:polyA, can also be included in this category.

[0249] The "Poly (A) tail" or "polyA tail" sequence can be composed of continuous adenines, such as a polynucleotide sequence composed of 120 consecutive adenines; it can also be a segmented polynucleotide sequence composed of one or more non-adenine spacer sequences in the continuous adenines, such as a polynucleotide sequence composed of 30 consecutive adenines, 3 to 20 non-adenines, 30 adenines, 3 to 20 non-adenines and 30 adenines.

[0250] "Upstream" and "downstream" in this application refer to the positions before and after (particularly immediately adjacent to) the target nucleic acid sequence in a certain transcription direction in the vector. When multiple promoters are present in the vector, thereby having multiple transcription directions, the target nucleic acid sequence can have multiple paired "upstream" and "downstream". In particular, the arrangement of the first terminator and the second terminator in this application makes it possible for the target nucleic acid sequence between the first terminator and the second terminator to not be transcribed along with the transcription behavior occurring outside the target nucleic acid sequence in the vector.

[0251] Mature mRNA has a long poly(A) tail, which plays a crucial role in maintaining mRNA stability, regulating mRNA translation efficiency, and maintaining mRNA transport. Currently, in vitro transcription is the most efficient method for bulk mRNA synthesis. There are two main approaches for poly(A) tailing in IVT. The first involves enzymatic synthesis. After mRNA is transcribed from a linear DNA template, poly(A) polymerase is added to catalyze the addition of AMP, converted from ATP, to the 3' end of the single-stranded RNA, forming the poly(A) tail. This method has the advantage of being simple, as the transcription template does not need to contain a poly(A) coding sequence. However, it has the limitation of unstable tail length. The second method involves co-transcription, where the poly(A) tail is directly transcribed from a pre-existing poly(A) tail coding sequence on a template plasmid DNA or PCR product. This approach eliminates the need for poly(A) polymerase, reduces process steps, saves costs, and ensures relatively stable tail length. However, in the preparation of upstream plasmids, it is necessary to reduce the loss of poly(A) tail coding sequences during the culture process, which is the key to providing high-yield, high-quality and uniform plasmid templates.

[0252] The inventors of this application have discovered in experiments that by i) increasing the distance between the replication origin and the poly(A) tail coding sequence in the vector, ii) adding terminators upstream and downstream of the poly(A) tail coding sequence or the mRNA expression cassette containing the poly(A) tail coding sequence, and / or iii) destroying the original lac promoter functional region in the vector, the amplification and / or expression (transcription) stability of the poly(A) tail coding sequence can be significantly improved, thereby increasing the purity of the plasmid in vitro transcription and / or the protein expression level of the output mRNA. In addition, by modifying the replication origin sequence of certain medium and low copy vectors, the vector yield can be further increased.

[0253] Therefore, the present application provides a vector or a vector backbone, which may comprise:

[0254] i) origin of replication, and

[0255] ii) a) a DNA expression cassette of interest comprising a promoter and a DNA sequence of interest, b) a DNA sequence of interest, c) a cloning site, d) a cloning site and a DNA sequence capable of forming a non-B-form DNA configuration, e) a DNA expression cassette comprising a promoter and a cloning site, or f) a DNA expression cassette comprising a promoter, a cloning site, and a DNA sequence capable of forming a non-B-form DNA configuration.

[0256] The vector or vector backbone comprising a), e), or f) in ii) can be used as a cloning vector or an expression vector. The target DNA sequence in a) can be any sequence, including sequences that form a B-form DNA structure and / or sequences that form a non-B-form DNA structure. The target DNA sequence can be inserted into the cloning site in e), and the target DNA sequence can include a DNA sequence that forms a non-B-form DNA structure. The cloning site in f) can be inserted into any sequence, including sequences that form a B-form DNA structure and / or sequences that form a non-B-form DNA structure.

[0257] The vector or vector backbone comprising b), or d) in ii) can be used solely as a cloning vector. The target DNA sequence in b) can be any sequence, including sequences that form a B-form DNA structure and / or sequences that form a non-B-form DNA structure. Any sequence can be inserted into the cloning site in d), including sequences that form a B-form DNA structure and / or sequences that form a non-B-form DNA structure.

[0258] In the vector backbone of ii) comprising c), depending on the sequence to be inserted into the cloning site, it can be used as a cloning vector or a cloning / expression vector. In the case where a promoter and a target DNA sequence are inserted into the cloning site, the vector can be used as a cloning / expression vector.

[0259] The origin of replication can be separated in the direction of replication by at least 150 bp, 200 bp, 300 bp, 500 bp, or 700 bp from a) the target DNA sequence in the target DNA expression cassette, b) the target DNA sequence, c) the cloning site, d) the DNA sequence capable of forming a non-B-form DNA configuration, e) the cloning site in the DNA expression cassette, or f) the DNA sequence capable of forming a non-B-form DNA configuration in the DNA expression cassette. The purpose of the separation of at least 150 bp, 200 bp, 300 bp, 500 bp, or 700 bp is to minimize the impact of the origin of replication on DNA sequences capable of forming a non-B-form DNA configuration, such as the coding sequence for the poly(A) tail, present in the vector during replication or transcriptional unwinding. DNA sequences capable of forming a non-B-form DNA configuration may form a non-B-form DNA configuration during DNA unwinding, thereby hindering replication or transcription and increasing mutations, such as deletions, in the vector. As the melting time increases, the probability of non-B-form DNA mutations increases. The melting time at the replication origin may be longer than elsewhere due to protein binding, etc. Therefore, increasing the distance between the replication origin and the DNA sequence that forms a non-B-form DNA configuration can reduce mutations in the DNA sequence that forms a non-B-form DNA configuration. The inventors of this application have experimentally discovered that by increasing the distance between the replication origin and the DNA sequence that forms a non-B-form DNA configuration from 100 bp to 247 bp, 500 bp, or even 700 bp, the proportion of vectors that retain an intact poly(A) tail encoding sequence during replication can be increased from 0.00% to 3.13%, 46.67%, or even 83.3%. The replication origin can enable the vector to replicate in a suitable host cell. Suitable host cells can be prokaryotes, such as Escherichia coli, or eukaryotic cells. In some embodiments, the replication origin can be a pUC replication origin, which can comprise, for example, the sequence of SEQ ID NO: 23.

[0260] In the case of, for example, a shuttle plasmid vector, the vector or vector backbone may further comprise a second origin of replication. Similarly, the second origin of replication may be spaced at least 150 bp, 200 bp, 300 bp, 500 bp, or 700 bp in the direction of replication from a) the target DNA sequence in the target DNA expression cassette, b) the target DNA sequence, c) the cloning site, d) the DNA sequence capable of forming a non-B-form DNA configuration, e) the cloning site in the DNA expression cassette, or f) the DNA sequence capable of forming a non-B-form DNA configuration in the DNA expression cassette. The second origin of replication may enable the vector to replicate in a suitable host cell, which may be a prokaryotic cell, such as Escherichia coli, or a eukaryotic cell.

[0261] Depending on the type of replication origin in the vector or vector backbone, the vector or vector backbone may also contain Rep protein expression elements, including Rep protein coding sequence and corresponding promoter, so that the vector or vector backbone becomes a broad host type.

[0262] The vector or vector backbone may further comprise a first terminator and a second terminator, located upstream and downstream of the sequences in ii), i.e., a) the target DNA expression cassette, b) the target DNA sequence, c) the cloning site, d) the cloning site and the DNA sequence capable of forming a non-B-form DNA configuration, and e) the DNA expression cassette, and arranged so that transcription in the vector or vector backbone outside of the above-mentioned sequences terminates at the upstream and downstream ends of these sequences. This design can greatly reduce melting within the region in ii), thereby maintaining the stability of the sequences in these regions, particularly the stability of DNA sequences capable of forming a non-B-form DNA configuration, such as the poly(A) tail encoding sequence. The inventors of this application have experimentally discovered that when the replication origin is spaced 247 bp from the specific sequence in ii), and terminators are added at both ends of the sequence in ii), the proportion of vectors that retain the intact poly(A) tail encoding sequence during replication can be increased from 3.13% to 37.14%. The first terminator may be a forward terminator, a bidirectional terminator, or a reverse terminator. The second terminator may be a forward terminator, a bidirectional terminator, or a reverse terminator. In some embodiments, the first terminator and the second terminator can all be bidirectional terminators." forward terminator " refers to the terminator that can make the transcription of terminator place chain terminate, " reverse terminator " refers to the terminator that can make the transcription on the complementary chain of terminator place chain terminate, and " bidirectional terminator " refers to the terminator that can make the transcription on terminator place chain and its complementary chain all terminate. Can be according to the concrete structure of carrier or carrier skeleton, for example the transcription direction of screening marker, to set terminator. Can use any terminator, as long as can guarantee that the transcription caused by the promoter outside the sequence region ii) in carrier or carrier skeleton terminates at the upstream and downstream of the sequence ii), makes that the transcription that occurs outside the sequence region ii) can not cause the melting of these regions. In universal carrier skeleton, bidirectional terminator can be set. In some embodiments, the first terminator can be rrnB T1 terminator and rrnB T2 terminator. In some embodiments, the second terminator can be lambda t0 terminator. The rrnB T1 terminator, rrnB T2 terminator, and lambda t0 terminator are known in the art and may comprise the nucleotide sequence shown in SEQ ID NO: 24, 25, or 26, respectively.

[0263] The cloning site in the vector or vector backbone can be a single cloning site or a multiple cloning site. The cloning site can be set according to actual needs.

[0264] The vector or vector backbone can also comprise a screening marker. The screening marker can be an antibiotic resistance gene, such as an ampicillin resistance gene, a tetracycline resistance gene, a chloramphenicol resistance gene or a kanamycin resistance gene. In particular, the screening marker is not present in the genome of the host cell to be transformed / transfected by the vector or vector backbone. In some embodiments, the screening marker is a kanamycin resistance gene. The vector or vector backbone can also comprise a promoter for the expression of the screening marker upstream of the screening marker. The screening marker may also promote replication and / or expression of the vector under certain circumstances, except that it can be used to confirm whether the vector is imported into the host cell. For example, when the vector or the vector backbone comprises the kanamycin resistance gene, and when the culture medium of the host cell contains kanamycin, the replication of the vector may be accelerated.

[0265] The vector or vector backbone can be circular. The vector or vector backbone can be a non-viral vector or a non-viral vector backbone. The vector or vector backbone can be a plasmid vector or a plasmid vector backbone. In some embodiments, the vector or vector backbone can be a circular plasmid vector or a circular vector backbone.

[0266] Given the leakiness of the lac promoter, particularly when the vector or vector backbone does not contain a fully functional lac promoter, the inventors have experimentally demonstrated that, after deactivating the lacP functional region, the proportion of vectors retaining the intact poly(A) tail encoding sequence during replication or passage can be increased from 31.25% to 50%. Even if the vector or vector backbone contains two terminators, inactivation of the lac promoter region is beneficial, as terminators cannot guarantee complete termination of transcription.

[0267] In some embodiments, the above-mentioned vector or vector backbone of the present application can be constructed to replicate and / or express mRNA and its poly (A) tail.

[0268] In addition to inserting the desired sequence into the cloning site, DNA sequences of interest, such as DNA sequences that can form non-B-form DNA conformations, specific mRNA coding sequences, promoters, etc., can also be added to the vectors or vector backbones of this application by methods such as homologous recombination. Vectors or vector backbones obtained by methods such as homologous recombination are also within the scope of protection of this application.

[0269] As a DNA delivery vector, recombinant adeno-associated virus (rAAV) has become the most important vector for genetic research and gene therapy due to its high safety, low immunogenicity, wide applicability, and long-lasting action. rAAV contains wild-type AAV ITRs or their derivatives at both ends, which are the only cis-acting elements in rAAV and are involved in rAAV replication, packaging, and exogenous gene expression.

[0270] However, the palindromic sequence of ITR forms a highly stable T-hairpin structure, and the high GC content makes it prone to mutation or recombination loss during bacterial reproduction. The importance of ITR integrity to the efficient replication and packaging of rAAV. Single-molecule real-time sequencing comprehensively analyzed the rAAV genome as a single complete molecule and found that in the HEK293 three-plasmid packaging system, the missing ITR was associated with the packaging of non-vector sequences. The impact of ITR deletion on rAAV virus packaging and the application of rAAV in gene therapy is complex and can produce unexpected biological consequences through vector-host interactions. If incomplete ITR sequences are used in the preclinical stage, the reproducibility of experiments within and across laboratories will be reduced, and may lead to experimental reproducibility when clinical trials are conducted using complete ITR sequences, delaying the transition to clinical applications. This problem is further exacerbated by the fact that partial loss of ITRs may go unnoticed due to their ability to self-repair during vector packaging in some cases (Wilmott, P., Lisowski, L., Alexander, IE, & Logan, GJ (2019). A user's guide to the inverted terminal repeats of adeno-associated virus. Human gene therapy methods, 30(6): 206-213).

[0271] Currently, there is no effective method to prevent ITR deletion during rAAV-ITR plasmid construction and E. coli amplification. Commercially available recombination-deficient strains such as JC8111, SURE2, Stabl3, and XL10-Gold are often used to construct and amplify ITR-containing plasmids, but this is not always effective.

[0272] The inventors of this application unexpectedly discovered that by i) extending the distance between the replication origin and the ITR sequence in the vector, ii) adding terminators upstream and downstream of the ITR, and / or iii) disrupting the original lac promoter region in the vector, the amplification and / or expression (transcription) stability of the ITR sequence can be significantly improved. Furthermore, these modifications to the vector can also increase vector yield and / or recombinant adeno-associated virus (rAAV) production, such as packaging titer.

[0273] Therefore, the present application provides a vector or a vector backbone, which may comprise:

[0274] i) origin of replication, and

[0275] ii) a) a target DNA expression cassette comprising, in sequence, a first ITR or a sequence derived therefrom, a promoter, a target DNA sequence, and a second ITR or a sequence derived therefrom, b) a target DNA sequence group comprising, in sequence, a first ITR or a sequence derived therefrom, a target DNA sequence, and a second ITR or a sequence derived therefrom, c) a DNA sequence group comprising, in sequence, a first ITR or a sequence derived therefrom, a cloning site, and a second ITR or a sequence derived therefrom, or d) a DNA expression cassette comprising, in sequence, a first ITR or a sequence derived therefrom, a promoter, a cloning site, and a second ITR or a sequence derived therefrom.

[0276] The vector or vector backbone comprising a) and d) in ii) can be used as a replication vector or, after AAV packaging, can be used to express a target DNA sequence. A target DNA sequence, such as an mRNA coding sequence or an mRNA coding sequence plus a poly(A) tail coding sequence, can be inserted into the cloning site in d). The target DNA sequence can be any sequence, including a DNA sequence that can form a B-form DNA configuration and / or a DNA sequence that can form a non-B-form DNA configuration.

[0277] The vector comprising b) in ii) can be used as a replication vector. The target DNA sequence can be any sequence, including a DNA sequence that can form a B-form DNA conformation and / or a DNA sequence that can form a non-B-form DNA conformation.

[0278] The vector backbone containing c) in ii) can be inserted into the cloning site of the target DNA sequence to form a cloning vector. The target DNA sequence can be any sequence, such as a DNA sequence that can form a B-type DNA configuration and / or a DNA sequence that can form a non-B-type DNA structure. A promoter and the target DNA sequence can also be inserted into the cloning site to form a cloning / expression vector. The target DNA sequence can be any sequence, such as a DNA sequence that can form a B-type DNA configuration and / or a DNA sequence that can form a non-B-type DNA structure.

[0279] The replication origin can be separated from a) the target DNA expression cassette, b) the target DNA sequence group, c) the DNA sequence group, or d) the DNA expression cassette by at least 150 bp, 200 bp, 300 bp, 500 bp, or 700 bp in the direction of replication. The purpose of this separation of at least 150 bp, 200 bp, 300 bp, 500 bp, or 700 bp is to minimize the impact of the replication origin on DNA sequences in the vector that can form non-B-form DNA structures, such as ITRs, during replication or transcriptional melting. ITRs may form hairpin or cruciform structures during DNA melting, hindering replication or transcription and increasing the likelihood of mutations, such as deletions. Prolonged melting times increase the likelihood of ITR mutations, and melting times at replication origins may be longer than at other locations due to protein binding. Therefore, increasing the distance between the replication origin and ITRs can reduce ITR mutations. If a) the target DNA expression cassette, b) the target DNA sequence group, c) the DNA sequence group, or d) the DNA expression cassette also contains other DNA sequences that can form a non-B-form DNA configuration, the mutation rate can be reduced by increasing the distance from the replication origin. The inventors have shown through experiments that when the replication origin and the selection marker in the vector are swapped, and the distance between the replication origin and the ITR in the replication direction is increased, the proportion of vector enzyme-digested bands caused by replication can be reduced from an average of 60.63% to 9.96%. In experiments in which the lacP functional region of the vector was deleted, it was also found that the closer the distance between lacP and the ITR, the greater the ITR sequence instability caused by, for example, transcription. This indirectly confirms the influence of the distance between the replication origin and the ITR on ITR sequence stability. The replication origin can enable the vector to replicate in a suitable host cell. The suitable host cell can be a prokaryotic cell, such as Escherichia coli, or a eukaryotic cell. In some embodiments, the replication origin can be a pUC replication origin, which can include, for example, the sequence of SEQ ID NO:23.

[0280] In the case of a shuttle plasmid vector, for example, the vector or vector backbone may further comprise a second origin of replication. Similarly, the second origin of replication may be spaced at least 150 bp, 200 bp, 300 bp, 500 bp, or 700 bp in the direction of replication from a) the target DNA expression cassette, b) the target DNA sequence group, c) the DNA sequence group, or d) the DNA expression cassette. The second origin of replication may allow the vector to replicate in a suitable host cell, which may be a prokaryotic cell such as Escherichia coli, or a eukaryotic cell.

[0281] Depending on the type of replication origin in the vector or vector backbone, the vector or vector backbone may also contain a Rep protein expression element, including a Rep protein coding sequence and a corresponding promoter, so that the vector becomes a broad host type.

[0282] The vector or vector backbone can also include a first terminator and a second terminator, which are positioned respectively at the upstream and downstream of a) target DNA expression cassette, b) target DNA sequence group, c) DNA sequence group, or d) DNA expression cassette, and are configured so that transcription outside a) target DNA expression cassette, b) target DNA sequence group, c) DNA sequence group, or d) DNA expression cassette in the vector or vector backbone terminates at the upstream and downstream of a) target DNA expression cassette, b) target DNA sequence group, c) DNA sequence group, or d) DNA expression cassette. Such a design can prevent the transcription occurring outside a) target DNA expression cassette, b) target DNA sequence group, c) DNA sequence group, or d) DNA expression cassette from causing melting within the a) target DNA expression cassette, b) target DNA sequence group, c) DNA sequence group, or d) DNA expression cassette region, thereby maintaining the stability of the sequence in this region, particularly the stability of the ITR. The inventors have experimentally discovered that when terminators are added to both ends of the ITR, the proportion of vector enzyme-digested impurities caused by replication can be further reduced from an average of 9.96% to 7.31%, or from 21.90% to 17.42%. The first terminator can be a forward terminator, a bidirectional terminator, or a reverse terminator. The second terminator can be a forward terminator, a bidirectional terminator, or a reverse terminator. In some embodiments, the first terminator and the second terminator can both be bidirectional terminators. The terminators can be set based on the specific structure of the vector, such as the transcription direction of the selection marker. Any terminator can be used as long as it can ensure that transcription outside of a) the target DNA expression cassette, b) the target DNA sequence group, c) the DNA sequence group, or d) the DNA expression cassette in the vector terminates upstream and downstream of the region, so that transcription occurring outside of the region does not cause melting within the region. Bidirectional terminators can be set in the universal vector backbone. In some embodiments, the first terminator can be the rrnB T1 terminator or the rrnB T2 terminator. In some embodiments, the second terminator can be the lambda t0 terminator. The rrnB T1 terminator, rrnB T2 terminator, and lambda t0 terminator are known in the art and may comprise the nucleotide sequence shown in SEQ ID NO: 24, 25, or 26, respectively.

[0283] The cloning site in the vector or vector backbone can be a single cloning site or a multiple cloning site. The cloning site can be set according to actual needs.

[0284] The vector or vector backbone can also comprise a screening marker. The screening marker can be an antibiotic resistance gene, such as an ampicillin resistance gene, a tetracycline resistance gene, a chloramphenicol resistance gene or a kanamycin resistance gene. Preferably, the screening marker is not present in the genome of the host cell to be transformed / transfected by the vector or vector backbone. In some embodiments, the screening marker is a kanamycin resistance gene. The vector or vector backbone can also comprise a promoter for the expression of the screening marker upstream of the screening marker. In addition to being used to confirm whether the vector is imported into the host cell, the screening marker may also promote the replication of the vector under certain circumstances. For example, when the vector or vector backbone comprises the kanamycin resistance gene, and when the culture medium of the host cell contains kanamycin, the replication of the vector may be accelerated.

[0285] The vector or vector backbone can be circular. The vector or vector backbone can be a non-viral vector or a non-viral vector backbone. The vector or vector backbone can be a plasmid vector or a plasmid vector backbone. In some embodiments, the vector or vector backbone can be a circular plasmid vector or a circular plasmid vector backbone.

[0286] Given the leakiness of the lac promoter, it is preferable that the vector or vector backbone not contain a fully functional lac promoter. Experimental studies have shown that deactivating the lacP functional region can increase the proportion of vectors retaining intact ITRs after replication, passage, or packaging from an average of 50.00% to 75.00%. Even if the vector or vector backbone contains two terminators, inactivation of the lac promoter region is beneficial, as terminators cannot guarantee 100% termination of transcription.

[0287] The first ITR or its derivative sequence and the second ITR or its derivative sequence may comprise the same or different nucleotide sequences. The first ITR and the second ITR may be wild-type AAV-ITR, comprising, for example, the nucleotide sequence shown in SEQ ID NO: 9. The first ITR derivative sequence and the second ITR derivative sequence may form a palindromic structure similar to that of a natural AAV-ITR, for example, comprising a palindromic arm and a palindromic stem, or only comprising a palindromic stem, and maintaining Rep protein binding ability. In some embodiments, the first ITR derivative sequence and the second ITR derivative sequence may comprise the nucleotide sequence shown in SEQ ID NO: 10, 11 or 12.

[0288] In some embodiments, the above-mentioned vector or vector backbone of the present application can be constructed to replicate and / or express a vector with ITR, or used to replicate and package rAAV vectors.

[0289] DNA sequences of interest, such as DNA sequences that can form non-B-form DNA structures, such as ITRs, can be added to the vectors or vector backbones of the present application by methods such as homologous recombination. Vectors or vector backbones obtained by methods such as homologous recombination are also within the scope of protection of the present application.

[0290] In this application, the inventors discovered and verified that i) lengthening the distance between the replication origin and the DNA sequence capable of forming a non-B-form DNA configuration, ii) adding terminators to both ends of the DNA sequence capable of forming a non-B-form DNA configuration or its expression cassette, and iii) removing the lacP functional region in the vector can significantly improve the replication and / or expression stability of the poly(A) tail encoding sequence and ITR sequence, and correspondingly increase the vector yield and / or in vitro transcription purity. The inventors have reason to believe that these modifications to the vector will also be beneficial to the replication and / or expression stability of other direct repeat sequences, inverted repeat sequences, and monopurine-monopyrimidine sequences.

[0291] According to the vector design of the present application, the vector can be prepared by de novo synthesis or the like.

[0292] In addition, existing vectors can also be modified based on the vector design of the present application. For example, in an existing vector or vector backbone, i) depending on the specific circumstances, the replication origin in the vector or vector backbone can be separated from the cloning site or the DNA sequence capable of forming a non-B-form DNA configuration by at least 150 bp, 200 bp, 300 bp, 500 bp, or 700 bp; ii) in the case of a lac promoter, the lac promoter can be inactivated; and / or iii) depending on the specific circumstances, terminators can be added upstream and downstream of the cloning site, the DNA sequence capable of forming a non-B-form DNA configuration, or the expression cassette of the DNA sequence capable of forming a non-B-form DNA configuration in the vector or vector backbone, respectively, so that transcription in the vector or vector backbone outside the above-mentioned regions is terminated upstream and downstream of these regions.

[0293] The vectors of the present application, including those prepared and modified, can be used to replicate and / or express target DNA sequences, particularly target DNA fragments containing direct repeats, single purine-single pyrimidine sequences, or inverted repeats. The direct repeats or single purine-single pyrimidine sequences can be, for example, poly(A) tail encoding sequences, polyA, polyG, polyC, or polyT. The inverted repeats can be, for example, ITRs or shRNA encoding sequences.

[0294] When using the vector of the present application to replicate and / or express the target DNA sequence, the conditions involved include transformation or transfection of the vector into host cells, culture of the host cells, in vitro transcription, etc. The corresponding conditions are known to those skilled in the art and can be adjusted according to actual conditions.

[0295] The vectors of the present application, including those prepared and those obtained by transformation, can be used to prepare recombinant adeno-associated virus vectors (rAAV). Specifically, i) a vector with a target DNA sequence group or a target DNA expression cassette with ITR at both ends of the present application can be provided, or a DNA sequence to be replicated and / or expressed can be inserted into the cloning site of the target DNA sequence group or the target DNA expression cassette with ITR at both ends of the present application to obtain a new vector; ii) optionally, these vectors are transformed / transfected into host cells, and the host cells are cultured under conditions that are conducive to vector replication; and iii) the vectors of i) or ii) are harvested, co-transformed / transfected into host cells with packaging plasmids and auxiliary plasmids, and the host cells are cultured under conditions that are conducive to vector packaging. If amplification is not required, step ii) can be omitted and three-plasmid packaging can be performed directly. The culture conditions of the host cells, the selection of packaging plasmids and auxiliary plasmids in the above steps are known to those skilled in the art and can be adjusted according to actual conditions. The packaging of adeno-associated virus vectors can also be carried out by methods other than three-plasmid packaging, as long as the purpose of vector packaging can be achieved.

[0296] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0297] Unless otherwise specified, the following sequences are all sequences on the positive strand of the vector.

[0298] Example 1: Adjusting the position of the replication origin (Ori) in the vector to improve the stability of the Poly(A) tail encoding sequence

[0299] Ori sequences are typically rich in adenine (A) and thymine (T). AT-rich DNA sequences are more susceptible to melting, and the melted Ori region provides space for the replication initiation protein to bind, thereby initiating plasmid replication. Furthermore, literature suggests that Ori elements may have initiation activity. When the 3' end of an Ori is adjacent to the poly(A) tail coding sequence, this coding sequence is highly susceptible to deletion or mutation during plasmid replication.

[0300] Take the pUC57-kana-T1 / T2 terminator vector (Nanjing GenScript, catalog number: C240PHC090-2) to construct a plasmid vector with an eGFP-120A-mRNA coding sequence (containing 120 consecutive adenines) inserted and different spacing distances between the 3' end of the Ori sequence and the poly(A) tail structure sequence to test the effect of the distance between the 3' end of the Ori sequence and the poly(A) tail structure sequence on the stability of the poly(A) tail structure sequence. The constructed vector map is shown in Figure 1, wherein the 3' end of the Ori sequence and the poly(A) tail structure sequence are separated by a 100bp, 247bp, 500bp, or 700bp spacer sequence (the sequences are shown in SEQ ID NO: 1-4, respectively, wherein the italic underlined sequences at both ends are sequences for homologous recombination with the pUC57-kana-T1 / T2 terminator vector, and the middle is a random sequence).

[0301] Specifically, 1.5 ng of the pUC57-kana-T1 / T2 terminator vector was taken, 5 μL of 10× cutsmart buffer, 2.5 μL of XhoI, and 2.5 μL of ApaLI were added, water was added to 50 μL, and the mixture was incubated at 37°C for 1 hour. The pUC57-kana-T1 / T2 terminator vector was digested with XhoI and ApaLI. The linearized vector product obtained was recovered using the AxyPrep-96 DNA gel recovery kit (Aixin Biotechnology Co., Ltd.). Afterwards, 0.8 ng of the recovered linearized vector was taken, 0.8 ng of each of the above-mentioned artificially synthesized spacer sequences (containing homology arm sequences homologous to the two ends of the vector, 30-50 bp at both ends), 10 μL of Genbuilder plus enzyme + buffer mix (Jiangsu GenScript Biotechnology Co., Ltd.), water was added to 20 μL, and the mixture was incubated at 50°C for 60 minutes to connect the spacer sequence fragment to the linearized vector fragment by homologous recombination. The obtained ligation product was used to transform Escherichia coli TOP10 competent cells and cultured on LB plates at 37°C for 12 h.

[0302] PCR screening was performed on the colonies. Specifically, the colony PCR reaction system (all reagents were from Jiangsu GenScript Biotechnology Co., Ltd.) contained 1 μL of forward primer, 1 μL of reverse primer, 1 μL of Taq DNA polymerase, 1 μL of 10× buffer, and 1 μL of dNTP. Water was added to 10 μL and the following program was performed in a PCR instrument (2720 Thermo Cycler): 95°C for 1 min; 95°C for 5 s, 55°C for 15 s, and 72°C for 1 min, 30 cycles; 72°C for 1 min; and storage at 4°C. For each recombinant plasmid vector, 48 clones with spacer sequences inserted were screened and inoculated into fresh 4 mL of LB liquid medium, respectively, and cultured with shaking at 37°C for 12 h.

[0303] Plasmids were extracted from the culture using the AxyPrep Plasmid DNA Mini Kit (Aixin Biotechnology Co., Ltd.), and the resulting plasmids were sequenced using the Sanger method on an Applied Biosystems 3730XL sequencer. Sequencing results were aligned and analyzed using SnapGene software. Positive clones were identified when the sequence aligned with the standard sequence and the poly(A) tail sequencing peak did not exceed 20% of the main peak. This was used to calculate the single-clone success rate for each recombinant plasmid vector construction, defined as the ratio of positive clones to the total number of selected clones. Figure 2 shows the sequencing peaks of positive clones (C, relatively pure poly(A) tail coding sequence with a sequencing peak below 20% of the main peak) and unsuccessful clones (A, missing poly(A) tail coding sequence; B, impure poly(A) tail coding sequence with a sequencing peak above 20% of the main peak). The results, as shown in Figure 3, show that when the distance between Ori 3' and the poly(A) tail coding sequence was 100 bp, the plasmid single cloning success rate was 0%, indicating that the sequencing purity of the poly(A) tail coding sequence of the plasmid was poor, with a high sequencing bottom peak. As the distance between Ori and the poly(A) tail coding sequence increased, the plasmid cloning success rate increased. When the distance between Ori and the poly(A) tail coding sequence was 700 bp, the plasmid single cloning success rate reached 83.33%, indicating that the sequencing purity of the poly(A) tail coding sequence was high, with the sequencing bottom peak less than 10% of the main poly(A) tail peak. This indicates that the farther the distance between Ori and the poly(A) tail coding sequence, the more stable the poly(A) tail coding sequence of the plasmid.

[0304] Example 2: Adding a terminator element to improve the stability of the poly (A) tail encoding sequence in the vector

[0305] The recombinant plasmid vector with a 247 bp spacer sequence constructed in Example 1 was taken, and the 247 bp spacer sequence in the plasmid was replaced with a 247 bp sequence containing a lambda t0 terminator (the sequence is shown in SEQ ID NO: 5, where the bold underlined sequence in the middle is the lambda t0 terminator, and the underlined italic parts at both ends are used for homologous recombination). The plasmid map is shown in Figure 4.

[0306] As described in Example 1, the obtained plasmid ligation product was used to transform Escherichia coli TOP10 competent cells and cultured on LB plates at 37°C for 12 h. Colonies were screened by PCR. The screened clones with terminator insertion were inoculated into 4 mL of fresh LB liquid culture medium and cultured with shaking at 37°C for 12 h. The cultured bacterial liquid was subjected to plasmid extraction, Sanger sequencing, and analysis.

[0307] The results, as shown in Figure 5 (AC), show that adding a terminator element between Ori and the poly(A) tail coding sequence increased the single cloning success rate from 3.13% to 37.14%. Compared to the 247bp spacer sequence without a terminator, plasmid sequencing showed lower peaks and higher purity of the poly(A) tail coding sequence. This suggests that the addition of a terminator element can improve the stability of the poly(A) tail coding sequence by, for example, blocking the influence of leaked promoter activity in the vector backbone on downstream functional regions.

[0308] Example 3: Deletion of the lac promoter functional region to improve the stability of the poly (A) tail coding sequence in the vector

[0309] A vector containing the eGFP-120A mRNA coding sequence was constructed as described in Example 1. In this unmodified pUC57-kana-T1 / T2 terminator vector, a 465-bp lac promoter region was located between the Ori and poly(A) tail coding sequences. This region, from the 5' to 3' direction, contained the CAP binding site, lac promoter, lactose operator, and M13 rev element. The single-clone success rate for the plasmid was 31.25%.

[0310] By base substitution, the sequence of the lac promoter functional region was changed to SEQ ID NO: 6, inactivating the functional region and constructing a new vector, the map of which is shown in Figure 6. Subsequently, clones with altered lac promoter functional region sequences were screened, cultured in LB medium with shaking, and plasmids were extracted and sequenced.

[0311] The results are shown in Figure 7. Inactivation of the lac promoter functional region increased the single cloning success rate from 31.25% to 50% and improved the stability of the poly(A) tail coding sequence in the plasmid vector.

[0312] Example 4: Construction of GS-MK, GS-PV and GS-CMV series vectors

[0313] According to the results of Examples 1, 2 and 3, an optimized vector was constructed.

[0314] Specifically, non-essential elements such as M13 rev, lac promoter, lactose operator, and CAP binding site were deleted from the pUC57-kana plasmid vector, T1 / T2 terminator and lambda t0 terminator elements were added upstream and downstream of the multiple cloning site, respectively, and the distance between the 3' end of Ori and the multiple cloning site was enlarged so that after a sequence such as an mRNA coding sequence was inserted into the multiple cloning site, the distance between the 3' end of Ori and the poly(A) coding sequence was greater than 700 bp.

[0315] In addition, synonymous mutations are performed on IIS sites such as BsaI, BspQI, BspMI, BpiI, and BsmbI on the vector backbone to remove these enzyme cleavage sites, and IIS enzyme recognition sites are added to the ends of the target DNA sequence to be inserted, such as the mRNA coding sequence and the poly(A) tail coding sequence. In this way, after the vector with the target DNA sequence inserted is linearized, the IIS type restriction endonuclease only recognizes outside the poly(A) tail coding sequence and cuts within the poly(A) coding sequence, thereby obtaining a relatively pure enzyme-cleaved fragment, and the polyT sequence in the enzyme-cleaved fragment will not be added to the non-T sequence, thereby obtaining a pure poly(A) tail. The pure poly(A) tail can maintain a higher protein expression level of the mRNA (Holtkamp S et al., (2006) Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells. Blood. 108(13): 4009-4017). This design also facilitates Golden Gate cloning of the vector.

[0316] The resulting vector, called the GS-MK vector, contains, from 5' to 3', the following: an origin of replication, rrnB T1 and rrnB T2 terminators, a multiple cloning site (MCS), a lambda t0 terminator, and a kanamycin resistance element. A plasmid map is shown in Figure 8.

[0317] Similarly, starting with the PVAX1 vector, the BsaI, BspQI, BspMI, and SpeI restriction sites on the synonymous mutation vector were removed, and T1 / T2 terminators and lambda t0 terminators were inserted upstream of the CMV promoter and downstream of the BGH poly(A) tail signal, respectively. The final constructed GS-PV vector contains, from 5' to 3', the following: a replication origin, rrnB T1 and rrnB T2 terminators, a CMV enhancer and promoter, a multiple cloning site, a bGH poly(A) signal, a lambda t0 terminator, and a kanamycin resistance element. A map is shown in Figure 9.

[0318] Furthermore, based on the GS-PV vector, a TC mutation in the replication origin of the original PVAX1 vector, which is different from the pUC replication origin, was corrected. The resulting vector, called GS-CMV, increases the plasmid copy number and reduces the risk of insufficient genome and yield during plasmid extraction.

[0319] Example 5: Functional testing of GS-MK, GS-PV and GS-CMV vectors

[0320] Various poly(A) tail coding sequences were inserted into the multiple cloning sites of GS-MK, GS-PV, and GS-CMV, including 100 adenines (100 A), 120 adenines (120 A), segmented 30 adenines + 30 adenines + 43 adenines (30 A + 3 non-A nucleotides + 30 A + 3 non-A nucleotides + 43 A), and segmented 31 adenines + 71 adenines (31 A + 8 non-A sequences + 71 A).

[0321] Specifically, the GS-MK vector was digested with NheI and ApaL1 to obtain a linearized vector. The gene fragment to be inserted was artificially synthesized, and the fragment contained from 5' to 3' direction: a multiple cloning site, an eGFP sequence (the function of this sequence is to increase the number of bases between Ori 3' and the poly (A) tail coding sequence so that the distance between the two is >700bp), the various poly (A) tail coding sequences mentioned above, and an IIS enzyme recognition site. The recombinant plasmid was obtained using the Gibson method. Taking GS-MK-100A-BspQI as an example, the inserted gene sequence is shown in SEQ ID NO: 27.

[0322] Similarly, the GS-PV or GS-CMV vector was digested with NheI and ApaL1 to obtain a linearized vector. The gene fragment to be inserted was synthesized, containing, from 5' to 3', a multiple cloning site, the poly(A) tail encoding sequences described above, and IIS enzyme recognition sites. Recombinant plasmids were obtained using the Gibson method. For GS-CMV-100A-BspQI, the inserted gene sequence is shown in SEQ ID NO:28.

[0323] As described above, the obtained plasmid with the poly(A) tail coding sequence inserted was used to transform Escherichia coli TOP10 competent cells and cultured on LB plates at 37°C for 12 hours; the colonies were screened by PCR; the screened clones with the poly(A) tail coding sequence inserted were inoculated into fresh 4 mL of LB liquid culture medium and cultured with shaking at 37°C for 12 hours; the cultured bacterial liquid was subjected to plasmid extraction, and Sanger sequencing and analysis were performed.

[0324] The sequencing peak graphs of the three vectors are shown in Figures 10 to 12. Overall, the bottom peak of plasmid sequencing is low and the purity of the Poly(A) tail encoding sequence is high.

[0325] Example 6: Comparison of GS-MK, GS-PV and GS-CMV vectors with commercial vectors

[0326] The eGFP-100A mRNA coding sequence was inserted into the GS-MK, GS-PV, and GS-CMV vectors constructed in Example 4, as well as the commercial Puc57-kana and PVAX1 vectors. The different vectors were analyzed and compared for single cloning success rate, poly(A) tail coding sequence stability, plasmid yield, in vitro transcription (IVT) purity, and mRNA eukaryotic protein expression level. The eGFP-100A mRNA coding sequence inserted into the above-mentioned vectors is shown in SEQ ID NO:29 and comprises a T7 promoter, a 5' UTR, an eGFP coding sequence, a 3' UTR, a poly(A) tail coding sequence, and a linearization restriction site.

[0327] According to the map, Puc57-kana contains LacZ but no terminator. The eGFP-100A mRNA coding sequence is inserted into the multiple cloning site, with a gap of approximately 400 bp between the poly(A) tail coding sequence and the 3' end of the replication origin. According to the map, the pVAX1 vector contains no terminator and the eGFP-100A mRNA coding sequence is inserted into the multiple cloning site, with a gap of approximately 700 bp between the poly(A) tail coding sequence and the 3' end of the replication origin.

[0328] The method for testing the stability of the poly(A) tail coding sequence is as follows: the plasmid is transformed into competent cells and then coated on a plate. A single clone is picked and inoculated into LB medium for culture at 37°C and 150 rpm min -1 After culturing for 12 h under the conditions of 1% LB (first generation bacterial solution), 100 μL of bacterial solution was transferred into 50 mL of LB medium for subculture and the mixture was incubated at 37°C and 150 rpm min -1 After culturing for 12 h under the conditions of 5% LB, 100 μL of the bacterial solution was transferred into 50 mL of LB medium for subculture at 37 ° C and 150 rpm min -1 After culturing for 12 h under the conditions of 5% paraformaldehyde and 1% paraformaldehyde (3rd generation bacterial solution), 2 mL of bacterial solution was taken for plasmid extraction and Sanger sequencing verification to obtain the probability of positive clones and the passage stability, that is, the ratio of the number of positive clones to the total number of picked plaque clones.

[0329] The plasmid yield was measured by transforming the plasmid into competent cells and then spreading the plasmid on a plate. A single colony was picked and inoculated into 400 mL of 3×LB medium. The culture medium was incubated at 37°C and 150 rpm / min. -1 The cells were shaken in an incubator for 13 h, the bacterial solution was collected, and the DNA yield of the plasmid was obtained by extraction.

[0330] IVT and purity testing are described below. Specifically, 100 μg of plasmid, 10 μL of BspQI enzyme, 30 μL of cutsmart buffer, and 300 μL of water were digested overnight at 37°C. The linearized product was recovered using an AxyPrep-96 DNA gel recovery kit. The plasmid linearized product was in vitro transcribed and purified according to the instructions of the mMessager mMachine T7 Ultra in vitro transcription kit. The purity of the IVT product was tested using an Agilent 5200 fragment analyzer.

[0331] Finally, the eukaryotic expression level of mRNA was tested. Specifically, 293T cells were inoculated and cultured in a 37°C, 5% CO2 incubator until the confluence reached 70-80%. Cell transfection was performed according to the instructions of the Lipofectamine 2000 kit. The transfection complex A solution contained 5μL Opit- and 0.5μL Lip2000, and the B solution contained 5μL Opit-MEM and 0.1μg mRNA. After mixing the A solution and the B solution, they were incubated at room temperature for 5 minutes. The mRNA product obtained from the above IVT was added to the cells and continued to be cultured in the incubator. 24h and 48h after the transfection was completed, the GFP fluorescence transfection effect was observed using an inverted fluorescence microscope, and the GFP fluorescence value was read using Image J software.

[0332] Experimental data showed that the monocloning success rate of GS-MK, GS-PV and GS-CMV vectors was significantly higher than that of pUC57-kana and PVAX1 vectors (Figure 13); the poly(A) tail coding sequence stability of the recombinant plasmids of GS-MK, GS-PV and GS-CMV vectors was significantly higher than that of pUC57-kana (Figure 14); under the same shake fermentation conditions, the recombinant plasmid yield of GS-MK and GS-CMV vectors was significantly higher than that of PVAX1 and GS-PV vectors (Figure 15); and because the GS-MK, GS-PV and GS-CMV vector plasmids have higher poly(A) tail coding sequence stability, their IVT purity and eukaryotic protein expression effect are also significantly better than pUC57-Kana (Figures 16 and 17).

[0333] Example 7: Deletion of the lac promoter functional region to improve the stability of the ITR structure in the vector

[0334] Four plasmids containing the lac promoter (lacP) functional region element (SEQ ID NO: 7) and AAV ITR were selected. The plasmid map is shown in FIG18 . The length of the ITR closest to lacP in the promoter direction is recorded in Table 1 .

[0335] The lac promoter sequence was replaced with SEQ ID NO: 8, and the lac operator, lac promoter, and CAP binding site in the lacP functional region were deleted to construct a new plasmid.

[0336] The plasmids before and after construction were transformed into the GenITRV1 strain (Nanjing GenScript) and cultured overnight at 37°C. Eight single clones were selected and cultured in LB at 37°C for 12 hours. The plasmids were extracted and treated with an ITR special kit (Nanjing GenScript) for Sanger sequencing. Clones without deletions or mutations in the ITR region were considered to pass Sanger sequencing, while those with deletions or mutations in the ITR region were considered to fail Sanger sequencing. The pass rates of Sanger sequencing for each plasmid before and after lacP deletion were calculated and summarized in Table 1.

[0337] Table 1. Sanger sequencing pass rate of plasmid clones before and after deletion of lacP element functional region

[0338] The results show that the closer the lacP element is to the ITR, the lower the Sanger sequencing pass rate, indicating that the lacP element has a certain impact on ITR stability. After deleting the lacP element, the average Sanger sequencing pass rate of the four plasmids increased from 50% to 75%, greatly improving the stability of the ITR structure.

[0339] Example 8: Design and construction of ITR stability vector

[0340] Transformation of pUC57 vector

[0341] The pUC57 plasmid used as a control in Example 6 is a commonly used molecular biology experimental vector, widely used in cloning expression, DNA sequencing, gene mutation, etc., and has the characteristics of high copy number, ease of operation, multiple cloning sites, and selectable marker genes. AAV-ITR shuttle plasmids carrying target genes designed based on the prokaryotic vector backbone of the pUC57 vector are widely used in AAV gene therapy. However, due to the specificity of the ITR sequence, its replication in E. coli is unstable and prone to deletion, requiring optimization and modification.

[0342] The prokaryotic vector backbone of pUC57 Kana (GenBank: LT671993.1) was taken and transformed. First, ITR sequences, namely wild-type AAV2-ITR sequences (SEQ ID NO: 9) or truncated AAV2-ITR sequences (SEQ ID NO: 10, 11, or 12), were added to both ends of the obtained vector, as shown in Figure 19 (A). Secondly, the position of pUC Ori was adjusted upstream of the KanR gene, and the distance between the end of pUC Ori and the front end of the 3'ITR sequence was lengthened to 1747bp. Again, the lac promoter in the plasmid was replaced with a nonsense sequence of the same length (SEQ ID NO: 13). The replacement sequence was derived from the natural plasmid of Escherichia coli and had no homology with the Escherichia coli genome or the pUC57 plasmid vector. The new vector backbone formed is called GenS-pUC57-AAV-NoTer.

[0343] Based on GenS-pUC57-AAV-NoTer, terminator sequences were added downstream of the 5'ITR position and upstream of the 3'ITR position, respectively, to obtain GenS-pUC57-AAV-Ter, as shown in FIG19(B).

[0344] Subsequently, the CMV enhancer, chicken β-actin promoter, EGFP, and bGH poly(A) signal were inserted between the 5'ITR and 3'ITR of the vectors GenS-pUC57-AAV-Ter and GenS-pUC57-AAV-NoTer. The IIS enzyme recognition sites on the vector backbone, such as BsaI, AarI, BpiI, BsmBI, and BspQI, were synonymously mutated to remove these restriction sites. These universal vectors GenS-pUC57-AAV-Noter-CMV (SEQ ID NO: 14, containing the ITR of SEQ ID NO: 9) and GenS-pUC57-AAV-Ter-CMV (SEQ ID NO: 15, containing the ITR of SEQ ID NO: 9) were prepared and assembled de novo by the gene synthesis department of GenScript Biopharmaceuticals.

[0345] Transformation of pSub201 vector

[0346] Samulski, the founder of AAV gene therapy, first described pSub201 (addgene.org / vector-database / 4231 / ) in October 1987 in Journal of Virology 61(10):pp.3096-3101. This vector contains the wild-type AAV-2 adeno-associated virus genome coding region and cis-terminal repeats (ITRs). This vector is an ideal choice for cloning because it is designed to perform restriction digestion with XbaI, maintain the integrity of the AAV-ITR in the plasmid backbone, remove the AAV genome coding region, and replace it with the gene of interest (GOI). Therefore, this vector was subsequently widely selected as a template for cloning for application in rAAV virus packaging and gene therapy, such as pAAV-tTA plasmid (GenBank:MZ708023.1). However, the prokaryotic backbone design of this vector has certain defects, which will cause the instability of the ITR structure, so certain modifications are needed to obtain a high-integrity ITR structure.

[0347] The prokaryotic vector backbone of the pAAV-tTA plasmid (GenBank: MZ708023.1) plasmid was taken ( Figure 20(A) ), and the phage-derived f1 ori was replaced with an additional sequence (SEQ ID NO: 16, excluding the portion homologous to the E. coli genome). The pUC Ori on the right was swapped with the ampicillin resistance gene. Elements such as the CMV enhancer, chicken β-actin promoter, EGFP, and bGH poly(A) signal were inserted between the 5' and 3' ITRs. The commonly used IIS enzyme recognition sites in the vector, such as BsaI, AarI, BpiI, BsmBI, and BspQI, and the restriction enzyme cleavage sites SmaI, AhdI, BglI, and BssHII, which are commonly used to verify the structural integrity of ITRs, were subjected to synonymous mutations. The resulting vector was named GenS-pSUB201-AAV-NoTer (SEQ ID NO: 17, containing the ITRs of SEQ ID NOs: 10 and 11). Next, terminator sequences were added downstream of the 5' ITR and upstream of the 3' ITR to generate the vector GenS-pSUB201-AAV-Ter (SEQ ID NO:18, containing ITRs of SEQ ID NOs:10 and 11), as shown in Figure 20(B). The distance between pUC Ori and the 3' ITR is 1814 bp. This newly designed vector was constructed and assembled de novo by GenScript Biopharmaceuticals' gene synthesis department.

[0348] Example 9: ITR structure stability test of pUC57 series vectors

[0349] Take pUC57 Kana (two ITRs have been added, i.e., before GenS-pUC57-AAV-NoTer adjusts the position of the Ori and KanR genes), GenS-pUC57-AAV-NoTer, and GenS-pUC57-AAV-Ter in Example 8, and insert gene fragment 1, gene fragment 2, gene fragment 3 or gene fragment 4 (as shown in SEQ ID NOs: 19-22, respectively) between the two ITRs through homologous recombination.

[0350] The resulting vector was cultured in 3xLB medium at 37°C under shake flask conditions for 16-18 hours. The plasmid was extracted and digested with SmaI. The SmaI cleavage site is located within the ITR; complete or partial deletion of the ITR sequence will result in digestion failure. The digestion products were subjected to agarose gel electrophoresis, with some results shown in Figure 21. Bands other than the target band are collectively referred to as "spurious bands." The percentage of these bands was analyzed using Gel Image System software (GIS). Specific values ​​are shown in Table 2.

[0351] Table 2. Proportion of SmaI enzyme-digested miscellaneous bands in pUC57-related vectors

[0352] The results show that the unmodified pUC57 Kana vector produced a high percentage of cloning artifacts, sometimes even reaching 100%. However, the optimized vector showed a significantly lower percentage of artifacts, indicating that optimization of the Ori position, terminator addition, and lacP deletion significantly improved the stability of the ITR structure. In particular, the GenS-pUC57-AAV-Ter vector, with terminators added to both ends of the ITR, showed a significantly lower percentage of artifacts than the GenS-pUC57-AAV-NoTer vector.

[0353] The extracted plasmids were then sequenced using an ITR sequencing kit (Nanjing GenScript). Sequencing results showed that sequencing of the unmodified pUC57 kana vector showed deletions, bottom peaks, double peaks, and unreachable sequences, resulting in a 0% Sanger sequencing pass rate. This was also frequently observed in other clones. However, sequencing peaks from the optimized vector showed no obvious bottom peaks, high ITR integrity, and a 100% Sanger sequencing pass rate.

[0354] The shake flask extraction yields of the three vectors were also tested. Specifically, clones containing correctly synthesized gene fragments from the three vectors were transformed into GenITRv1 competent cells. After overnight incubation at 37°C, single clones were selected and placed in a test tube containing 4 mL of LB medium. The cells were shaken at 30°C and 180 rpm for 16 hours. 400 μL of the cultured seed solution was then transferred to a 400 mL flask containing 3× LB medium and shaken at 37°C and 200 rpm for 13 hours. The cells were then harvested by centrifugation, lysed using alkaline lysis, and precipitated with isopropanol. Finally, the plasmid precipitate was dissolved in TE, and the plasmid concentration was measured using a UV spectrophotometer. The results are summarized in Table 3. As can be seen, the yields of the two optimized vectors increased by 44% and 26%, respectively, compared to the unmodified pUC57 Kana vector.

[0355] Table 3. Shake flask extraction yields of pUC57-related vectors (μg)

[0356] Furthermore, the effects of the three vectors on subsequent AAV packaging were evaluated. Specifically, a three-plasmid packaging system was used, with three vectors (containing inserted gene 1) as shuttle plasmids, and the packaging plasmids pRC2-mi342 (Takara, Catalog No.: 6230) / pRC-DJ (Cell Biolabs INC, Catalog No. VPK-420-DJ-8) of AAV2 and AAVDJ, as well as the auxiliary plasmid pHelper (Takara, Catalog No.: 6230), were used respectively. The industrial standard for shuttle plasmids is 1.5 μg / ml. The shuttle plasmid, packaging plasmid, and auxiliary plasmid were added to the AAV-MAX transfection kit (ThermoFisher, A50515 / A50516) at a molar ratio of 2:4:1, and virus packaging was performed according to the instructions of the kit. Reference Viral titer was determined using the Titration Kit (using real-time PCR) (TaKaRa, Cat. No. 6233) version 2, as described in the kit instructions, to evaluate the titer of AAV viral packaging. The results are summarized in Table 4.

[0357] Table 4. Virus packaging titers of three vectors

[0358] As shown in Table 4, the optimized vectors achieved higher viral packaging titers than the unmodified pUC57 Kana vector, and the GenS-pUC57-AAV-Ter vector achieved higher titers than the GenS-pUC57-AAV-NoTer vector. This is consistent with the aforementioned SmaI digestion results, which showed that the GenS-pUC57-AAV-Ter vector had a lower proportion of SmaI-digested artifacts and higher ITR integrity.

[0359] Example 10: ITR structure stability test of pSub201 series vectors

[0360] Four gene segments (genes 1-4) identical to those in Example 8 were cloned using pAAV-tTA, GenS-pSUB201-AAV-NoTer, and GenS-pSUB201-AAV-Ter from Example 8. Specifically, gene segment 1, gene segment 2, gene segment 3, or gene segment 4 was inserted between two ITRs through homologous recombination.

[0361] As described in Example 9, the vectors were transformed into GenITRv1 competent cells, and single colonies containing the plasmid were selected for shake flask fermentation and plasmid extraction. The yields of the three vectors are shown in Table 5. As can be seen, the modified GenS-pSUB201-AAV-NoTer and GenS-pSUB201-AAV-Ter vectors showed average yield increases of 66% and 35%, respectively, after shake flask fermentation, compared to the unmodified pAAV-tTA vector.

[0362] Table 5. Yields of three vectors after shake flask fermentation (μg)

[0363] As described in Example 9, the three vectors were digested with SmaI and subjected to agarose gel electrophoresis. Bands other than the target band were collectively referred to as miscellaneous bands. GIS was used to analyze the proportion of miscellaneous bands after SmaI digestion. The specific values ​​are shown in Table 6. It can be seen that the unmodified pAAV-tTA vector has a high proportion of miscellaneous bands, while the proportion of miscellaneous bands in the modified GenS-pSUB201-AAV-NoTer and GenS-pSUB201-AAV-Ter vectors is significantly reduced, and the stability of the ITR structure is significantly improved.

[0364] Table 6. Proportion of SmaI enzyme-digested miscellaneous bands using three vectors

[0365] As described in Example 9, a three-plasmid packaging system was used, with three vectors (with gene 1 inserted) as shuttle plasmids, and AAV2 packaging plasmid pRC2-mi342 and helper plasmid pHelper (Takara, Catalog No. 6230). The industrial standard for shuttle plasmids was 1.5 μg / ml. The shuttle plasmid, packaging plasmid, and helper plasmid were added to the AAV-MAX transfection kit (ThermoFisher, A50515 / A50516) at a molar ratio of 2:4:1, and virus packaging was performed according to the kit instructions. Viral titers were measured using the Titration Kit (using real-time PCR) (TaKaRa, Cat. No. 6233), version 2, according to the kit instructions, to evaluate the titer of AAV viral packaging. The results are shown in Table 7. Both GenS-pSUB201-AAV-NoTer and GenS-pSUB201-AAV-Ter increased rAAV viral titers by 44% compared to pAAV-tTA.

[0366] Table 7. Virus packaging titers of three vectors

[0367] The sequences involved in the application are as follows.

[0368] Although the present application has been described in conjunction with one or more embodiments, it should be understood that the present application is not limited to these embodiments. The description in this application is intended to cover all variants and equivalents, all of which are included in the subject matter and scope of the appended claims. All documents cited in this article are incorporated herein by reference in their entirety.

Claims

1. A carrier comprising: i) origin of replication, ii) a first terminator, iii) a) a target DNA expression cassette comprising a promoter and a target DNA sequence, wherein the target DNA sequence comprises a DNA sequence capable of forming a non-B-form DNA configuration, or b) a target DNA sequence comprising a DNA sequence capable of forming a non-B-form DNA configuration, iv) a second terminator, and v) a selection marker and a promoter controlling the expression of the selection marker, in, The first terminator and the second terminator are respectively located upstream and downstream of the target DNA expression cassette of a), and are configured to terminate transcription outside the target DNA expression cassette of a) upstream and downstream of the target DNA expression cassette of a); or The first terminator and the second terminator are respectively located upstream and downstream of the target DNA sequence of b), and are configured to terminate transcription outside the target DNA sequence of b) upstream and downstream of the target DNA sequence of b).

2. The vector of claim 1, wherein the replication origin is spaced at least 150 bp in the replication direction from a) the DNA sequence capable of forming a non-B-form DNA configuration in the target DNA expression cassette or b) the DNA sequence capable of forming a non-B-form DNA configuration in the target DNA sequence.

3. The vector of claim 2, wherein the replication origin is spaced at least 500 bp in the replication direction from a) the DNA sequence capable of forming a non-B-form DNA configuration in the target DNA expression cassette or b) the DNA sequence capable of forming a non-B-form DNA configuration in the target DNA sequence.

4. The vector according to any one of claims 1 to 3, wherein the DNA sequence capable of forming a non-B-form DNA configuration is a direct repeat sequence, an inverted repeat sequence, or a monopurine-monopyrimidine sequence.

5. The vector of claim 4, wherein the DNA sequence capable of forming a non-B-form DNA configuration is a poly(A) tail encoding sequence.

6. The vector according to any one of claims 1 to 5, wherein the replication origin is a pMB1 replication origin, a pBR322 replication origin, a ColE1 replication origin, a R6K replication origin, a p15A replication origin, a pSC101 replication origin, a pUC replication origin, a pET replication origin or a derivative thereof.

7. The vector of any one of claims 1 to 6, wherein the first terminator is selected from one or more of rrnB T1 terminator, rrnB T2 terminator, lambda t0 terminator, TonB terminator, Amp terminator, T7 terminator and CYC1 terminator; the second terminator is selected from one or more of rrnB T1 terminator, rrnB T2 terminator, lambda t0 terminator, TonB terminator, Amp terminator, T7 terminator and CYC1 terminator.

8. The vector according to any one of claims 1 to 7, wherein the selection marker is an ampicillin resistance gene, a tetracycline resistance gene, a chloramphenicol resistance gene, a kanamycin resistance gene, or a streptomycin resistance gene.

9. The vector according to any one of claims 1 to 8, wherein the target DNA sequence in the target DNA expression cassette of a) further comprises a cloning site, and the target DNA sequence of b) further comprises a cloning site.

10. The vector according to any one of claims 1 to 9, wherein the target DNA expression cassette further comprises an enhancer and / or a transcription terminator.

11. The vector according to claim 10, comprising in order: pUC replication origin, rrnB T1 terminator and rrnB T2 terminator, CMV enhancer, CMV promoter, target DNA sequence, bGH poly (A) signal, lambda t0 terminator, and kanamycin resistance element; or pUC origin of replication, rrnB T1 terminator and rrnB T2 terminator, target DNA sequence, lambda t0 terminator, and kanamycin resistance element.

12. A carrier comprising: i) origin of replication, ii) a first terminator, iii) a) a target DNA expression cassette, which sequentially comprises a first ITR or a sequence derived therefrom, a promoter, a target DNA sequence, and a second ITR or a sequence derived therefrom, or b) a target DNA sequence group, which sequentially comprises a first ITR or a sequence derived therefrom, a target DNA sequence, and a second ITR or a sequence derived therefrom, iv) a second terminator, and v) a selection marker and a promoter controlling the expression of the selection marker, in, The first terminator and the second terminator are respectively located upstream and downstream of the target DNA expression cassette of a), and are configured to terminate transcription outside the target DNA expression cassette of a) upstream and downstream of the target DNA expression cassette of a); or The first terminator and the second terminator are respectively located upstream and downstream of the target DNA sequence group b), and are configured to terminate transcription outside the target DNA sequence group b) upstream and downstream of the target DNA sequence group b).

13. The vector according to claim 12, wherein the replication origin is spaced at least 150 bp from a) the target DNA expression cassette or b) the target DNA sequence group in the replication direction, preferably, at least 500 bp.

14. The vector of claim 12 or 13, wherein the first terminator is selected from one or more of rrnB T1 terminator, rrnB T2 terminator, lambda t0 terminator, TonB terminator, Amp terminator, T7 terminator and CYC1 terminator, and the second terminator is selected from one or more of rrnB T1 terminator, rrnB T2 terminator, lambda t0 terminator, TonB terminator, Amp terminator, T7 terminator and CYC1 terminator.

15. The vector according to any one of claims 12 to 14, wherein the replication origin is a pMB1 replication origin, a pBR322 replication origin, a ColE1 replication origin, a R6K replication origin, a p15A replication origin, a pSC101 replication origin, a pUC replication origin, a pET replication origin or a derivative thereof.

16. The vector according to any one of claims 12 to 15, wherein the selection marker is an ampicillin resistance gene, a tetracycline resistance gene, a chloramphenicol resistance gene, a kanamycin resistance gene, or a streptomycin resistance gene.

17. The vector according to any one of claims 12 to 16, wherein the first ITR or a sequence derived therefrom comprises the nucleotide sequence shown in SEQ ID NO:9, 10, 11 or 12, and the second ITR or a sequence derived therefrom comprises the nucleotide sequence shown in SEQ ID NO:9, 10, 11 or 12.

18. The vector according to any one of claims 12 to 17, wherein the target DNA expression cassette further comprises an enhancer and / or a transcription terminator.

19. The vector of claim 18, comprising, in order: pUC replication origin, rrnB T1 terminator and rrnB T2 terminator, first ITR or its derivative sequence, CMV enhancer, chicken β-actin promoter, target DNA sequence, bGH poly (A) signal, second ITR or its derivative sequence, lambda t0 terminator, and kanamycin resistance element.

20. A method for modifying a vector, comprising: i) providing a vector comprising: a) the origin of replication, b) bi) cloning site, b-ii) a DNA expression cassette comprising a promoter and a cloning site, b-iii) a DNA expression cassette comprising a promoter, a cloning site and a DNA sequence capable of forming a non-B-form DNA configuration, b-iv) a target DNA sequence, which comprises a DNA sequence capable of forming a non-B-form DNA conformation, bv) a target DNA expression cassette comprising a promoter and a target DNA sequence, wherein the target DNA sequence comprises a DNA sequence capable of forming a non-B-form DNA configuration, b-vi) a DNA sequence group, which sequentially comprises a first ITR or a sequence derived therefrom, a cloning site, and a second ITR or a sequence derived therefrom, b-vii) a DNA expression cassette comprising, in order, a first ITR or a sequence derived therefrom, a promoter, a cloning site, and a second ITR or a sequence derived therefrom, b-viii) a target DNA expression cassette comprising, in order, a first ITR or a sequence derived therefrom, a promoter, a target DNA sequence, and a second ITR or a sequence derived therefrom, or b-ix) a target DNA sequence group, which sequentially comprises a first ITR or a derivative sequence thereof, a target DNA sequence, and a second ITR or a derivative sequence thereof, and c) a selection marker and a promoter for controlling the expression of the selection marker, and ii) adding a first terminator and a second terminator to the vector so that they are located upstream and downstream of the sequence in b), respectively, and arranged so that transcription other than the sequence in b) in the vector is terminated upstream and downstream of the sequence in b).

21. The method of claim 20, further comprising spacing the replication origin by at least 150 bp, preferably at least 500 bp, in the replication direction from bi) the cloning site, b-ii) the cloning site in the DNA expression cassette, b-iii) the DNA sequence in the DNA expression cassette capable of forming a non-B-type DNA configuration, b-iv) the DNA sequence in the target DNA sequence capable of forming a non-B-type DNA configuration, bv) the DNA sequence in the target DNA expression cassette capable of forming a non-B-type DNA configuration, b-vi) a DNA sequence group, b-vii) a DNA expression cassette, b-viii) a target DNA expression cassette, or b-ix) a target DNA sequence group.

22. The method of claim 20 or 21, wherein the vector further comprises a lac promoter, the method further comprising inactivating the lac promoter.

23. A composition comprising the vector of any one of claims 1-11, the vector of any one of claims 12-19, or the vector obtained by the method of any one of claims 20-22.

24. A method for replicating a target DNA sequence, comprising: i) providing a vector according to any one of claims 1 to 11, or a vector according to any one of claims 12 to 19; ii) transforming / transfecting the vector of step i) into a host cell; and iii) culturing the host cells under conditions conducive to replication of the vector.

25. A method for expressing a target DNA sequence, comprising: i) providing a vector comprising a) a target DNA expression cassette according to any one of claims 1 to 11, and ii) transforming / transfecting the vector of step i) into a host cell and culturing the host cell under conditions conducive to the expression of the target DNA sequence, or linearizing the vector of step i) and performing in vitro transcription; or i) providing a vector comprising a) a target DNA expression cassette according to any one of claims 12 to 19, ii) transforming / transfecting the vector of step i) into a host cell, and culturing the host cell under conditions conducive to the expression of the target DNA sequence, Linearizing the vector of step i) and performing in vitro transcription; or The vector of step i) is co-transformed / transfected with a packaging plasmid and a helper plasmid into a host cell, the host cell is cultured under conditions conducive to vector packaging, the packaged vector is recovered, the host cell is transformed / transfected, and the host cell is cultured under conditions conducive to the purpose The host cells are cultured under conditions that allow the target DNA to be expressed.

26. The method of claim 25, further comprising, between step i) and step ii), transforming / transfecting the vector of step i) into a host cell, and culturing the host cell under conditions conducive to vector replication.

27. A method for preparing a recombinant adeno-associated virus vector, comprising: i) providing a vector according to any one of claims 12 to 19; ii) transforming / transfecting the vector of step i) into a host cell; iii) culturing the host cell under conditions conducive to replication of the vector; and iv) harvesting the vector from the host cells in step iii), and co-transforming / transfecting the vector with a packaging plasmid and a helper plasmid into the host cells, and culturing the host cells under conditions conducive to vector packaging.

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