Method for improving production efficiency of adeno-associated virus

Abstract

The present disclosure provides a polynucleotide comprising: an adeno-associated virus (AAV) 5'inverted terminal repeat (ITR) sequence, a recombinant adeno-associated virus (rAAV) transgene cassette sequence, an AAV 3 'ITR sequence, and one or both of an insulator sequence linked to the 5' ITR sequence, or an inverted sequence thereof, and an insulator sequence linked to the 3 'ITR sequence, or an inverted sequence thereof. The disclosure also provides vectors and cells comprising the polynucleotides, as well as methods of producing rAAV vectors using the polynucleotides and the vectors.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the field of viral vectors, in particular to adeno-associated virus (AAV) vectors, and more particularly to methods for improving the efficiency of production of recombinant AAV vectors. BACKGROUND

[0002] Adeno-associated virus (AAV) belongs to the parvovirus family, which consists of a protein capsid and a single-stranded DNA genome of about 4.7 kb. Both ends of the AAV genome are two T-shaped inverted terminal repeat (ITR) sequences, which are mainly used as a viral replication origin and a packaging signal (see Naso MF et al., Adeno-Associated Virus (AAV) as a Vector for Gene Therapy. BioDrugs. 2017 Aug; 31(4): 317-334, which is incorporated herein by reference).

[0003] AAV is a naturally defective virus, and its productive replication requires the help of a helper virus. In the absence of a helper virus, the wild-type AAV genome can either latently exist in the form of an episome in the host cell or integrate into the host cell genome. The AAVS1 (Adeno-Associated Virus integration Site 1) region on human chromosome 19 (AC010327.8 (7774-11429), see https: / / www.ncbi.nlm.nih.gov / gene / 17) is the preferred site for AAV DNA integration. Integration at the AAVS1 site requires the ITR of AAV, Rep78 / 68 and the AAV p5 promoter. Rep68 / 78 is able to bind to the Rep Binding Site (RBS) in AAVS1 as well as in the AAV ITR to form a complex between the AAV ITR and AAVS1, and is able to introduce a nick at the Terminal Resolution site (TRS) in AAVS1 to integrate the AAV DNA into the genome.

[0004] Recombinant AAV (rAAV) is a commonly used vector in which the native coding (e.g., Rep and Cap genes) and non-coding sequences between the ITR sequences are replaced with a transgene cassette containing the foreign gene of interest. Currently, transient transfection is the most commonly used production method for rAAV. rAAV can be produced in HEK293 cells by transient transfection of three plasmids: a pHelper plasmid carrying adenoviral E2A, E4, and VA RNA genes, a Rep-Cap plasmid carrying AAV Rep and Cap genes, and a transgene plasmid carrying the rAAV transgene cassette (see Samulski RJ, et al. AAV-Mediated Gene Therapy for Research and Therapeutic Purposes. Annu Rev Virol. 2014 Nov; 1(1): 427-51, which is incorporated herein by reference). This system requires transfection of a large number of plasmids to make high titer virus and causes trouble for downstream purification. In addition, AAV can be produced by co-infecting two baculoviruses expressing Rep-Cap and gene of interest (GOI), respectively, into insect cells. However, these baculoviruses are unstable at higher passages and are time-consuming to make. HeLa packaging cells with stably integrated Rep-Cap and GOI have also been developed. However, this system still requires wild-type adenovirus as a helper virus, which poses a risk of contaminating replicative adenovirus in the AAV product. Therefore, there is an urgent need in the art for cell lines and related methods for producing AAV that can be easily scaled up to large-scale production to provide reproducible and stable results while limiting contamination and reducing costs.

[0005] Insulators on chromosomes are usually located in the non-coding regions of the genome, which are boundary DNA elements involved in regulating gene transcription. Insulators can specifically regulate the transcription of genes by forming protein complexes with transcription factors and their co-factors, matrix proteins, or chromatin modification proteins, etc. The functions of insulators mainly include: 1) enhancer blocking: an enhancer is a remote regulatory element that up-regulates gene expression, when an insulator is located between the enhancer and the promoter, it can prevent the enhancer from enhancing the activity of gene transcription; 2) heterochromatin barrier: heterochromatin is densely packed and folded chromatin, the genes located in this region are almost not transcribed, while the insulator can hinder the spread of the heterochromatin structure, thereby ensuring that the genes in the genomic region protected by it can be normally expressed.

[0006] Currently, there is no report of inserting an insulator sequence into a transgene vector carrying a gene of interest (GOI) for preparing an AAV vector to improve the production efficiency of the AAV vector. SUMMARY

[0007] To solve the technical problem of improving the production efficiency of recombinant AAV vectors, the present disclosure provides the following.

[0008] In one aspect, the present disclosure provides a polynucleotide (hereinafter referred to as "polynucleotide of the present disclosure") comprising: an adeno-associated virus (AAV) 5' inverted terminal repeat (ITR) sequence, a recombinant adeno-associated virus (rAAV) transgene cassette sequence, an AAV 3' ITR sequence, and one or both of an insulator sequence or its reverse sequence linked to the 5' ITR sequence and an insulator sequence or its reverse sequence linked to the 3' ITR sequence.

[0009] In one embodiment, in the polynucleotide of the present disclosure, the insulator sequence or its reverse sequence linked to the 5' ITR sequence is linked to the 5' end of the 5' ITR sequence.

[0010] In one embodiment, in the polynucleotide of the present disclosure, the insulator sequence or its reverse sequence linked to the 3' ITR sequence is linked to the 3' end of the 3' ITR sequence.

[0011] In one embodiment, in the polynucleotide of the present disclosure, the insulator sequence is derived from the AAVS1 region of human chromosome 19.

[0012] In one embodiment, in the polynucleotide of the present disclosure, the insulator sequence comprises or is derived from a DNase 1 hypersensitive site in the AAVS1 region of human chromosome 19.

[0013] In one embodiment, in the polynucleotide of the present disclosure, the insulator is an HS4 insulator.

[0014] In another aspect, the present disclosure provides a vector (hereinafter referred to as "vector of the present disclosure") comprising the polynucleotide of the present disclosure.

[0015] In another aspect, the present disclosure provides a cell (hereinafter referred to as "cell of the present disclosure") comprising the polynucleotide of the present disclosure or the vector of the present disclosure.

[0016] In another aspect, the present disclosure provides use of the polynucleotide of the present disclosure, the vector of the present disclosure, or the cell of the present disclosure in the preparation of an rAAV vector.

[0017] In another aspect, the present disclosure provides a method for preparing an rAAV vector (hereinafter referred to as "method for preparing an rAAV vector of the present disclosure") comprising introducing the polynucleotide of the present disclosure or the vector of the present disclosure into a cell.

[0018] In one embodiment, in the method for producing an rAAV vector of the present disclosure, the polynucleotide of the present disclosure or the vector of the present disclosure is transiently transfected into the cell.

[0019] In one embodiment, in the method for producing an rAAV vector of the present disclosure, the polynucleotide of the present disclosure is integrated into the genome of the cell.

[0020] The production efficiency of the rAAV vector can be significantly improved by adding an insulator sequence at one or both sides of the recombinant AAV ITR sequence. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Titer results of rAAV vector production according to one embodiment of the present disclosure are shown.

[0022] Figure 2 Titer results of rAAV vector production according to another embodiment of the present disclosure are shown. DETAILED DESCRIPTION

[0023] The following examples are used to illustrate the technical solutions of the present application, and the following examples should not be considered as limiting the scope and spirit of the present application.

[0024] In one aspect, the present disclosure provides a polynucleotide (hereinafter referred to as "polynucleotide of the present disclosure") comprising: an adeno-associated virus (AAV) 5' inverted terminal repeat (ITR) sequence, a recombinant adeno-associated virus (rAAV) transgene cassette sequence, an AAV 3' ITR sequence, and one or both of an insulator sequence or its reverse sequence linked to the 5' ITR sequence and an insulator sequence or its reverse sequence linked to the 3' ITR sequence. As used in the present disclosure, the term "insulator" has the meaning commonly understood by one of skill in the art. An insulator is a cis-regulatory sequence that is capable of blocking an enhancer from activating a target promoter or acting as a barrier to impede the spread of heterochromatin structure. Whether a sequence has insulator function can be determined by routine means in the art, e.g., computational analysis and experimental validation. The computational analysis can be performed, e.g., by searching for known insulator motifs using tools such as CADD or FIMO, analyzing the position of the sequence relative to a gene, analyzing the chromatin features around the sequence. The experimental validation can be performed, e.g., by reporter assays, chromatin conformation capture (3C), transgene assays, functional genomics assays, etc. As used in the present disclosure, the term "insulator sequence" can include an insulator sequence and the complement of the insulator sequence. As used in the present disclosure, the term "reverse sequence thereof" or "reverse sequence of the insulator sequence" includes the reverse sequence of the insulator sequence and the reverse sequence of the complement of the insulator sequence. As used in the present disclosure, the term "recombinant adeno-associated virus transgene cassette" or "rAAV transgene cassette" refers to a transgene cassette comprising a foreign gene of interest and its regulatory sequences for replacing the original coding sequences (such as Rep gene sequences and Cap gene sequences) and non-coding sequences between the 5' ITR sequence and the 3' ITR sequence of AAV used in the preparation of rAAV vectors.

[0025] In one embodiment, in the polynucleotide of the present disclosure, the insulator sequence or its reverse sequence linked to the 5' ITR sequence is linked to the 5' end of the 5' ITR sequence. In one example, the insulator sequence or its reverse sequence is directly linked to the 5' end of the 5' ITR sequence. In one example, the insulator sequence or its reverse sequence is spaced apart from the 5' end of the 5' ITR sequence by, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases. The number of bases spaced apart between the insulator sequence or its reverse sequence and the 5' end of the 5' ITR sequence is not particularly limited as long as the linkage of the insulator sequence to the ITR sequence is capable of achieving the same or similar effects as the present disclosure.

[0026] In one embodiment, in the polynucleotide of the present disclosure, the insulator sequence or its reverse sequence linked to the 3' ITR sequence is linked to the 3' end of the 3' ITR sequence. In one example, the insulator sequence or its reverse sequence is directly linked to the 3' end of the 3' ITR sequence. In one example, the insulator sequence or its reverse sequence is spaced apart from the 3' end of the 3' ITR sequence by, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases. The number of bases spaced apart between the insulator sequence or its reverse sequence and the 3' end of the 3' ITR sequence is not particularly limited as long as the connection of the insulator sequence to the ITR sequence can achieve the same or similar effects as the present disclosure.

[0027] In the present disclosure, the insulator sequence that can be used in connection with the ITR sequence is not particularly limited, and can be an insulator sequence commonly used in the art, for example, a gypsy insulator sequence of the Drosophila melanogaster gypsy transposon, an Idefix insulator sequence of the Idefix retrotransposon, a homie insulator sequence, a scs / scs' insulator sequence, SF1 and SF2 insulator sequences, a chicken beta globin HS4 insulator sequence, a BX-C Fab-8 insulator sequence, and the like, or a derivative sequence thereof.

[0028] In the present disclosure, the AAV ITR sequence that can be used is not particularly limited, and can be derived from various naturally occurring serotypes of AAV, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, and the like, or various variants or derivatives thereof, and can also be derived from various artificially constructed serotypes of AAV.

[0029] In one embodiment, in the polynucleotide of the disclosure, the insulator sequence is derived from the AAVS1 region of human chromosome 19. As used in the present disclosure, the term "AAVS1 region of human chromosome 19" refers to the AAVS1 (Adeno-Associated Virus integration Site 1) region located on human chromosome 19 (AC010327.8 (7774-11429), see https: / / www.ncbi.nlm.nih.gov / gene / 17). It has been reported that an insulator exists near the DNase 1-hypersensitive site (DHS-S1) in the AAVS1 region, which is capable of having blocking function on enhancer and preventing the spread of heterochromatin (see Toshihiko Ogata et al., Identification of an Insulator in AAVS1, a Preferred Region for Integration of Adeno-Associated Virus DNA. J Virol. 2003 Aug;77(16):9000-9007, which is incorporated herein by reference). In one embodiment, in the polynucleotide of the disclosure, the insulator sequence comprises or is derived from the DNase 1-hypersensitive site (DHS-S1) in the AAVS1 region of human chromosome 19. In one example, the insulator can comprise the sequence of SEQ ID NO: 20 or a sequence having at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50% sequence identity to SEQ ID NO: 20, as long as the sequence maintains the function of the insulator.

[0030] In one embodiment, in the polynucleotide of the present disclosure, the insulator is a HS4 insulator. Chicken hypersensitive site 4 (cHS4, also referred to herein as HS4 insulator or HS4I) is one of the most characterized and most commonly used insulators in the art. There is 16 kb of constitutive heterochromatin upstream of HS4. In red blood cells, if this heterochromatin structure spreads to the globin gene region, globin will not be normally expressed and thus red blood cells will function abnormally. HS4 can prevent the spread of this dense chromatin structure, thereby ensuring normal expression of the globin gene (see Rincon-Arano et al., Protection against telomeric position effects by the chicken cHS4 beta-globin insulator. Proc Natl Acad Sci U S A. 2007 Aug 28; 104(35), which is incorporated herein by reference). The HS4 insulator has enhancer-blocking function (prevents interaction between enhancer and promoter) (see Chung J H et al., Characterization of the chicken beta-globin insulator. Proc Natl Acad Sci USA, 1997, 94(2): 575-580, which is incorporated herein by reference).

[0031] In another aspect, the present disclosure provides a vector (hereinafter referred to as “vector of the present disclosure”) comprising the polynucleotide of the present disclosure. The vector that can be used as the vector of the present disclosure is not particularly limited, and can be a vector commonly used in the art for delivering a nucleic acid of interest. In one example, the vector is a non-viral vector. In one example, the vector is a plasmid vector. In one example, the vector is a lipid nanoparticle (LNP) or a liposome. In one example, the vector is a viral vector, such as a lentiviral vector, etc.

[0032] In another aspect, the present disclosure provides a cell (hereinafter referred to as "the cell of the present disclosure") comprising the polynucleotide of the present disclosure or the vector of the present disclosure. The origin of the cell of the present disclosure is not particularly limited. In one example, the cell is a eukaryotic cell. In one example, the cell is an insect cell. In one example, the cell is a mammalian cell. In one example, the cell can be, for example, selected from the group consisting of 293T cells, HepG2 cells, CHO cells, BHK cells, HEK293 cells, COS cells, NIH / 3T3 cells, Hela cells, A549 cells, Vero cells, HT1080 cells, Te671 cells, CEM cells, NSO cells, PerC6 cells, and the like, and a cell derived therefrom. In one aspect, the cell is a HEK293 cell or a cell derived therefrom. In one aspect, the host cell is a 293T cell or a cell derived therefrom.

[0033] In another aspect, the present disclosure provides use of the polynucleotide of the present disclosure, the vector of the present disclosure, or the cell of the present disclosure in the preparation of an rAAV vector.

[0034] In another aspect, the present disclosure provides a method for preparing an rAAV vector (hereinafter referred to as "the method for preparing an rAAV vector of the present disclosure") comprising introducing the polynucleotide of the present disclosure or the vector of the present disclosure into a cell. In the present disclosure, the cell that can be used in the method of the present disclosure is not particularly limited. In one example, the cell is a eukaryotic cell. In one example, the cell is an insect cell. In one example, the cell is a mammalian cell. In one example, the cell can be, for example, selected from the group consisting of 293T cells, HepG2 cells, CHO cells, BHK cells, HEK293 cells, COS cells, NIH / 3T3 cells, Hela cells, A549 cells, Vero cells, HT1080 cells, Te671 cells, CEM cells, NSO cells, PerC6 cells, and the like, and a cell derived therefrom. In one aspect, the cell is a HEK293 cell or a cell derived therefrom. In one aspect, the host cell is a 293T cell or a cell derived therefrom.

[0035] In one embodiment, in the method for preparing an rAAV vector of the present disclosure, the polynucleotide of the present disclosure or the vector of the present disclosure is transiently introduced into the cell. The method for transiently introducing the polynucleotide of the present disclosure or the vector of the present disclosure into a cell is not particularly limited. The polynucleotide of the present disclosure or the vector of the present disclosure can be transiently introduced into a cell by various conventional methods in the art.

[0036] In one embodiment, in the method for preparing an rAAV vector of the present disclosure, the polynucleotide of the present disclosure is integrated into the genome of the cell. The method for integrating the polynucleotide of the present disclosure into the genome of the cell is not particularly limited. The polynucleotide of the present disclosure can be integrated into the genome of the cell by various conventional methods in the art. Methods for integrating a nucleic acid fragment of interest into the genome of a cell are known in the art, including but not limited to, transposon system-mediated integration methods, viral vector-mediated integration methods, gene editing system-mediated integration methods, site-specific recombinase-mediated integration methods, and linearized template-mediated integration methods.

[0037] In the following examples, various plasmids were constructed by adding (or not adding) various insulator sequences in different ways at the ITRs (in the following examples, the ITRs of AAV2 were used as examples) on both sides of the rAAV transgene cassette containing a GOI (Luciferase-IRES-EGFP-WPRE was used as an example in the following examples):

[0038] An AAVS1 sequence (DHS-S1 sequence, SEQ ID NO: 20) was added at the 5' end of the 5' ITR, so that the resulting sequence had the form of AAVS1-ITR(5')-GOI-ITR(3') (where "GOI" represents an rAAV expression cassette containing a GOI (same below), the ITR(5') sequence and the ITR(3') sequence can be represented by SEQ ID NO: 18 and SEQ ID NO: 19, respectively, and the sequence of AAVS1-ITR(5') can be represented by SEQ ID NO: 22) (plasmid 06.01.1997, the group of plasmids used in the following examples and figures is referred to as "5'A");

[0039] An AAVS1(R) sequence (SEQ ID NO: 21) was added at the 3' end of the 3' ITR, so that the resulting sequence had the form of ITR(5')-GOI-ITR(3') -AAVS1(R) (the sequence of ITR(3') -AAVS1(R) can be represented by SEQ ID NO: 23) (plasmid 06.01.1998, the group of plasmids used in the following examples and figures is referred to as "3'A(R)");

[0040] An AAVS1 sequence and an AAVS1(R) sequence were added at the 5' end of the 5' ITR and at the 3' end of the 3' ITR, respectively, so that the resulting sequence had the form of AAVS1-ITR(5')-GOI-ITR(3') -AAVS1(R) (plasmid 06.01.2028, the group of plasmids used in the following examples and figures is referred to as "5'A-3'A(R)");

[0041] AAVS1 (R) sequence at the 5' end of the 5' ITR such that the resulting sequence has the form AAVS1 (R)-ITR(5')-GOI-ITR(3') (the sequence of AAVS1 (R)-ITR(5') can be as set forth in SEQ ID NO: 24) (plasmid 06.01.2727, the group of plasmids used in the following examples and figures using this plasmid is denoted as "5'A(R)");

[0042] AAVS1 sequence at the 3' end of the 3' ITR such that the resulting sequence has the form ITR(5')-GOI-ITR(3')-AAVS1 (the sequence of ITR(3')-AAVS1 can be as set forth in SEQ ID NO: 25) (plasmid 06.01.2728, the group of plasmids used in the following examples and figures using this plasmid is denoted as "3'A");

[0043] AAVS1 (R) sequence at the 5' end of the 5' ITR and AAVS1 sequence at the 3' end of the 3' ITR such that the resulting sequence has the form AAVS1 (R)-ITR(5')-GOI-ITR(3')-AAVS1 (plasmid 06.01.1999, the group of plasmids used in the following examples and figures using this plasmid is denoted as "5'A(R)-3'A");

[0044] AAVS1 sequence at the 5' end of the 5' ITR and AAVS1 sequence at the 3' end of the 3' ITR such that the resulting sequence has the form AAVS1-ITR(5')-GOI-ITR(3')-AAVS1 (plasmid 06.01.2905, the group of plasmids used in the following examples and figures using this plasmid is denoted as "5'A-3'A");

[0045] AAVS1 (R) sequence at the 5' end of the 5' ITR and AAVS1 (R) sequence at the 3' end of the 3' ITR such that the resulting sequence has the form AAVS1 (R)-ITR(5')-GOI-ITR(3')-AAVS1 (R) (plasmid 06.01.2904, the group of plasmids used in the following examples and figures using this plasmid is denoted as "5'A(R)-3'A(R)");

[0046] HS4I sequence (SEQ ID NO: 26) at the 5' end of the 5' ITR such that the resulting sequence has the form HS4I-ITR(5')-GOI-ITR(3') (the sequence of HS4I-ITR(5') can be as set forth in SEQ ID NO: 28) (plasmid 06.01.2729, the group of plasmids used in the following examples and figures using this plasmid is denoted as "5'H");

[0047] An HS4I(R) sequence (SEQ ID NO: 27) was added to the 3' end of the 3' ITR such that the resulting sequence had the form ITR(5')-GOI-ITR(3')-HS4I(R) (the sequence of ITR(3')-HS4I(R) can be set forth as SEQ ID NO: 29) (plasmid 06.01.2730, the group of plasmids used in the following examples and figures using this plasmid is denoted "3'H(R)");

[0048] An HS4I sequence and an HS4I(R) sequence were added to the 5' end of the 5' ITR and to the 3' end of the 3' ITR, respectively, such that the resulting sequence had the form HS4I-ITR(5')-GOI-ITR(3')-HS4I(R) (plasmid 06.01.2027, the group of plasmids used in the following examples and figures using this plasmid is denoted "5'H-3'H(R)").

[0049] An HS4I(R) sequence was added to the 5' end of the 5' ITR such that the resulting sequence had the form HS4I(R)-ITR(5')-GOI-ITR(3') (the sequence of HS4I(R)-ITR(5') can be set forth as SEQ ID NO: 30) (plasmid 06.01.2731, the group of plasmids used in the following examples and figures using this plasmid is denoted "5'H(R)").

[0050] An HS4I sequence was added to the 3' end of the 3' ITR such that the resulting sequence had the form ITR(5')-GOI-ITR(3')-HS4I (the sequence of ITR(3')-HS4I can be set forth as SEQ ID NO: 31) (plasmid 06.01.2732, the group of plasmids used in the following examples and figures using this plasmid is denoted "3'H").

[0051] An HS4I(R) sequence and an HS4I sequence were added to the 5' end of the 5' ITR and to the 3' end of the 3' ITR, respectively, such that the resulting sequence had the form HS4I(R)-ITR(5')-GOI-ITR(3')-HS4I (plasmid 06.01.2733, the group of plasmids used in the following examples and figures using this plasmid is denoted "5'H(R)-3'H)").

[0052] An HS4I sequence was added to the 5' end of the 5' ITR and to the 3' end of the 3' ITR, such that the resulting sequence had the form HS4I-ITR(5')-GOI-ITR(3')-HS4I (plasmid 06.01.2903, the group of plasmids used in the following examples and figures using this plasmid is denoted "5'H-3'H").

[0053] HS4I(R) sequence at the 5' end of the 5' ITR and at the 3' end of the 3' ITR, so that the resulting sequence has the form HS4I(R)-ITR(5')-GOI-ITR(3')-HS4I(R) (plasmid 06.01.2902, the group of this plasmid is denoted "5'H(R)-3'H(R)" in the following examples and figures);

[0054] No insulator sequence was added at the 5' end of the 5' ITR and at the 3' end of the 3' ITR (plasmid 06.01.2026, the group of this plasmid is denoted "ORI" in the following examples and figures).

[0055] Example 1 : Plasmid construction method

[0056] The molecular cloning techniques used in the following examples, such as amplification of DNA fragments, restriction endonuclease digestion of DNA fragments, gel recovery of DNA fragments, ligation of DNA fragments, transformation of competent cells with ligation products, plasmid extraction and identification, etc. are all maturely known techniques in the art. The following examples involve the following reagents, materials or services: high-fidelity DNA polymerase (Agilent); restriction endonuclease (NEB); T4 ligase (Thermo); DNA fragment gel recovery kit (Omega, D2500-02); plasmid extraction kit (OMEGA, D6943-02); chemically competent cells (XL-10 gold); 06.01.0007 (14BF007_pHelper, Fenghui Bio, BR417); nucleotide sequences represented by SEQ ID NO:1-SEQ ID NO:17 and plasmid 06.01.2014 (pmkSBT3-2xHS4I-AAV(R2C2)-hPGK(M3)-BSD, sequence represented by SEQ ID NO:32), plasmid 06.01.2018 (sequence represented by SEQ ID NO:33) and plasmid 06.01.1274 (pmk-MCS, sequence represented by SEQ ID NO:34) were synthesized by GenScript. Table 1 is an element composition description of sequences SEQ ID NO:1-SEQ ID NO:31. Table 2 is a description of each functional element in the plasmid. The functional elements used in each plasmid involved in the following examples are examples for realizing the present disclosure, and those skilled in the art can expect that replacing the sequences of each functional element used in the following examples with other biologically functional similar element sequences can also achieve the same or similar effects as described in the present disclosure, including but not limited to plasmid backbone (such as replication origin, resistance gene, etc.), restriction enzyme site, transposon recognition sequence, inducible system response element, promoter, intron, polyadenylation signal (PolyA), different codon-optimized gene sequences, mutants of the sequences of each functional element and gene sequence, and cloning sites, cloning order and cloning direction of the sequences of each functional element and gene sequence. The specific plasmid construction method is as follows:

[0057] Construction of 06.01.2028: Synthetic sequence SEQ ID NO:1 was double-digested with SbfI and AscI, and ligated to the corresponding site of synthetic plasmid 06.01.2018 also treated with enzymes, to obtain plasmid 06.01.2028.

[0058] Construction of 06.01.1997: Synthetic sequence SEQ ID NO:2 was double-digested with SbfI and AscI, and ligated to the corresponding site of synthetic plasmid 06.01.2018 also treated with enzymes, to obtain plasmid 06.01.1997.

[0059] Construction 06.01.1998: The synthetic sequence SEQ ID NO: 3 was double digested with Sbf I and Asc I and ligated into the corresponding sites of the synthetic plasmid 06.01.2018, which was also treated with the enzymes, to give the plasmid 06.01.1998.

[0060] Construction 06.01.1999: The synthetic sequence SEQ ID NO: 4 was double digested with Sbf I and Asc I and ligated into the corresponding sites of the synthetic plasmid 06.01.2018, which was also treated with the enzymes, to give the plasmid 06.01.1999.

[0061] Construction 06.01.2727: The synthetic sequence SEQ ID NO: 5 was double digested with Sbf I and Asc I and ligated into the corresponding sites of the synthetic plasmid 06.01.2018, which was also treated with the enzymes, to give the plasmid 06.01.2727.

[0062] Construction 06.01.2728: The synthetic sequence SEQ ID NO: 6 was double digested with Sbf I and Asc I and ligated into the corresponding sites of the synthetic plasmid 06.01.2018, which was also treated with the enzymes, to give the plasmid 06.01.2728.

[0063] Construction 06.01.2904: The synthetic sequence SEQ ID NO: 7 was double digested with Sbf I and Asc I and ligated into the corresponding sites of the synthetic plasmid 06.01.2018, which was also treated with the enzymes, to give the plasmid 06.01.2904.

[0064] Construction 06.01.2905: The synthetic sequence SEQ ID NO: 8 was double digested with Sbf I and Asc I and ligated into the corresponding sites of the synthetic plasmid 06.01.2018, which was also treated with the enzymes, to give the plasmid 06.01.2905.

[0065] Construction 06.01.2026: The synthetic sequence SEQ ID NO: 9 was double digested with Sbf I and Asc I and ligated into the corresponding sites of the synthetic plasmid 06.01.2018, which was also treated with the enzymes, to give the plasmid 06.01.2026.

[0066] Construction 06.01.2027: The synthetic sequence SEQ ID NO: 10 was double digested with Sbf I and Asc I and ligated into the corresponding sites of the synthetic plasmid 06.01.2018, which was also treated with the enzymes, to give the plasmid 06.01.2027.

[0067] Construction 06.01.2730: Synthetic sequence SEQ ID NO: 12 was double digested with Sbf I and Asc I and ligated into the corresponding sites of the same enzyme treated synthetic plasmid 06.01.2018 to yield plasmid 06.01.2730.

[0068] Construction 06.01.2730: Synthetic sequence SEQ ID NO: 12 was double digested with Sbf I and Asc I and ligated into the corresponding sites of the same enzyme treated synthetic plasmid 06.01.2018 to yield plasmid 06.01.2730.

[0069] Construction 06.01.2731: Synthetic sequence SEQ ID NO: 13 was double digested with Sbf I and Asc I and ligated into the corresponding sites of the same enzyme treated synthetic plasmid 06.01.2018 to yield plasmid 06.01.2731.

[0070] Construction 06.01.2732: Synthetic sequence SEQ ID NO: 14 was double digested with Sbf I and Asc I and ligated into the corresponding sites of the same enzyme treated synthetic plasmid 06.01.2018 to yield plasmid 06.01.2732.

[0071] Construction 06.01.2733: Synthetic sequence SEQ ID NO: 15 was double digested with Sbf I and Asc I and ligated into the corresponding sites of the same enzyme treated synthetic plasmid 06.01.2018 to yield plasmid 06.01.2733.

[0072] Construction 06.01.2902: Synthetic sequence SEQ ID NO: 16 was double digested with Sbf I and Asc I and ligated into the corresponding sites of the same enzyme treated synthetic plasmid 06.01.2018 to yield plasmid 06.01.2902.

[0073] Construction 06.01.2903: Synthetic sequence SEQ ID NO: 17 was double digested with Sbf I and Asc I and ligated into the corresponding sites of the same enzyme treated synthetic plasmid 06.01.2018 to yield plasmid 06.01.2903.

[0074] Table 1. Sequence element composition description

[0075] SEQ ID NO: Sequence element composition description 1 SbfI-AAVS1-ITR-EF1α-luciferase-IRES-EGFP-WPRE-ITR-AAVS1(R)-AscI 2 SbfI-AAVS1-ITR-EF1α-luciferase-IRES-EGFP-WPRE-ITR-AscI 3 SbfI-ITR-EF1α-luciferase-IRES-EGFP-WPRE-ITR-AAVS1(R)-AscI 4 SbfI-AAVS1(R)-ITR-EF1α-luciferase-IRES-EGFP-WPRE-ITR-AAVS1-AscI 5 SbfI-AAVS1(R)-ITR-EF1α-luciferase-IRES-EGFP-WPRE-ITR-AscI 6 SbfI-ITR-EF1α-luciferase-IRES-EGFP-WPRE-ITR-AAVS1-AscI 7 SbfI-AAVS1(R)-ITR-EF1α-luciferase-IRES-EGFP-WPRE-ITR-AAVS1(R)-AscI 8 SbfI-AAVS1-ITR-EF1α-luciferase-IRES-EGFP-WPRE-ITR-AAVS1-AscI 9 SbfI-ITR-EF1α-luciferase-IRES-EGFP-WPRE-ITR-AscI 10 SbfI-HS4I-ITR-EF1α-luciferase-IRES-EGFP-WPRE-ITR-HS4I(R)-AscI 11 SbfI-HS4I-ITR-EF1α-luciferase-IRES-EGFP-WPRE-ITR-AscI 12 SbfI-ITR-EF1α-luciferase-IRES-EGFP-WPRE-ITR-HS4I(R)-AscI 13 SbfI-HS4I(R)-ITR-EF1α-luciferase-IRES-EGFP-WPRE-ITR-AscI 14 SbfI-ITR-EF1α-luciferase-IRES-EGFP-WPRE-ITR-HS4I-AscI 15 SbfI-HS4I(R)-ITR-EF1α-luciferase-IRES-EGFP-WPRE-ITR-HS4I-AscI 16 SbfI-HS4I(R)-ITR-EF1α-luciferase-IRES-EGFP-WPRE-ITR-HS4I(R)-AscI 17 SbfI-HS4I-ITR(5')-EF1a-luciferase-IRES-EGFP-WPRE-ITR(3')-HS4I-AscI 18 ITR(5') 19 ITR(3') 20 AAVS1 21 AAVS1(R) 22 AAVS1-ITR(5') 23 ITR(3')-AAVS1(R) 24 AAVS1(R)-ITR(5') 25 ITR(3')-AAVS1 26 HS4I 27 HS4I(R) 28 HS4I-ITR(5') 29 ITR(3')-HS4I(R) 30 HS4I(R)-ITR(5') 31 ITR(3')-HS4I

[0076] Table 2. Plasmid functional element description

[0077]

[0078]

[0079] Example 2: Toxin production test in case of transient transfection of transgene plasmid

[0080] In this example, rAAV was produced by transient transfection of 293T cells with Rep-Cap plasmid (plasmid 06.01.2014) carrying AAV2 Rep and Cap genes, pHelper plasmid (plasmid 06.01.0007) carrying adenovirus E2A, E4 and VA RNA genes, and various transgene plasmids constructed by adding (or not adding) various insulator sequences in different ways at the ITRs flanking the rAAV transgene cassette containing GOI as described above. The effect of adding (or not adding) various insulator sequences on viral production titer was compared.

[0081] Specifically, 293T (ATCC-CRL3216) cells were cultured at 37°C, 5% CO2, with DMEM (Sigma, D6429) complete medium supplemented with 10% FBS (ExCell, 1H116). 293T cells were seeded in 6-well plates (Corning, 3516) at 8E5 cells / well. After 24 hours of culture, 200 μl of transfection reagent containing PEI was added to each well, which contained a total plasmid amount of 5 μg, with a molar ratio of Rep-Cap plasmid: pHelper plasmid: transgene plasmid of 1:1:1, and a mass ratio of plasmid to PEI MAX (Polysciences, 24765-1) of 1:4. After 3 hours of transfection, the culture medium was replaced with new DMEM complete medium. After 48 hours of transfection, an equal volume of 1% Tween 20 was added to each well, followed by shaking for 15 minutes, then centrifugation at 3000 rpm for 10 minutes to harvest the virus solution, and detection of viral genome copy number (vg) using a qPCR instrument (ABI, A28574).

[0082] Figure 1 In this example, NC is the negative control group transiently transfected with empty vector plasmid (06.01.1274) only. The results show that the vg values of the 5'A group, 3'A(R) group, 5'A-3'A(R) group, 5'A(R) group, 3'A group, 5'A(R)-3'A group, 5'A-3'A group, 5'A(R)-3'A(R) group, 5'H group, 3'H(R) group, 5'H-3'H(R) group, 5'H(R) group, 3'H group, 5'H(R)-3'H group, 5'H-3'H group and 5'H(R)-3'H(R) group are significantly improved compared with the ORI group. The specific results are shown in Table 3.

[0083] The results show that, in the case of transient transfection of plasmids, the rAAV vector production efficiency of each group with different insulator sequences added at the ITR is significantly improved compared to the ORI group without any insulator sequence added at the ITR.

[0084] Table 3. Transgene plasmid transient transfection toxicity test results (vg)

[0085]

[0086] Example 3: Toxicity test in the case of stable insertion of a transgene cassette into the genome

[0087] In this example, rAAV transgene cassettes in the various transgene plasmids constructed as described above and their flanking ITRs (and various insulator sequences added, if any) were stably inserted into the genome of 293T cells, and then Rep-Cap plasmid (plasmid 06.01.2014) and pHelper plasmid (plasmid 06.01.0007) were transiently transfected to produce rAAV vectors. The effect of adding (or not adding) various insulator sequences on viral production titer was compared in the case of stable expression.

[0088] First, the rAAV transgene cassettes in the various transgene plasmids constructed with PB transposase recognition sequences (ITR sequences of the PB transposon system, see the plasmid construction section of Example 1) flanking both sides and their flanking ITRs (and various insulator sequences added, if any) were efficiently integrated into the cell genome using the PB transposon system, and the cells were selected using the resistance gene (puromycin resistance) carried by the vector. Specifically, 293T cells were seeded at 8E5 cells / dish in a 60 mm dish containing 3 ml of DMEM complete medium. After 24 hours of culture at 37°C, 5% CO2, 500 μl of calcium phosphate transfection reagent containing 0.12 mol / L calcium chloride, IX HEPES buffer, and 4 μg of total plasmid amount were added per dish. The medium was replaced with DMEM complete medium 6 hours after transfection. The medium was replaced with selection medium containing 1 μg / ml puromycin 24 hours after transfection. All cells were trypsinized 48 hours after transfection and seeded in 100 mm dishes (Corning, 430167), and continuously selected under this drug pressure for at least 3 generations until the cell line was stable. After the cells grew stably, the drug was removed and the cells were transferred to DMEM complete medium.

[0089] After that, the stable cells described above were transiently transfected with Rep-Cap plasmid and pHelper plasmid. Specifically, the various stable cells described above were inoculated into 6-well plates at 8E5 cells / well, and the culture volume was 2 ml. After 24 hours of culture, transfection was performed according to the PEI method, and 200 μl of transfection mixture containing a total plasmid amount of 5 μg was added to each well, in which the plasmid amount of Rep-Cap plasmid and pHelper plasmid was 3.5 μg, the molar ratio of Rep-Cap plasmid:pHelper plasmid was 1:1, and the plasmid amount of empty vector plasmid (06.01.1274) was 1.5 μg, and the mass ratio of plasmid to PEI MAX was 1:4. Three hours after transfection, the culture medium was replaced with DMEM complete medium. Forty-eight hours after transfection, an equal volume of 1% Tween 20 was added to lyse the cells in the 6-well plates, the plates were placed on a shaker at 600 rpm at room temperature for 15 min, and then the virus supernatant was collected by centrifugation at 3000 rpm at room temperature for 5 min, and the viral genome copy number (vg) was detected using a qPCR instrument.

[0090] Figure 2 Among them, the NC group is a negative control group that is only transiently transfected with an empty vector plasmid (06.01.1274). Compared with the ORI group, the vg values of the 5'A group, the 3'A(R) group, the 5'A-3'A(R) group, the 5'A(R) group, the 3'A group, the 5'A(R)-3'A group, the 5'A-3'A group, the 5'A(R)-3'A(R) group, the 5'H group, the 3'H(R) group, the 5'H-3'H(R) group, the 5'H(R) group, the 3'H group, the 5'H(R)-3'H group, the 5'H-3'H group, and the 5'H(R)-3'H(R) group are significantly improved. The specific results are shown in Table 4.

[0091] This result shows that, in the case of stable insertion of the transgene cassette into the genome, the production efficiency of the rAAV vector of each group in which various insulator sequences are added in different ways at the ITR is significantly improved compared with the ORI group in which no insulator sequence is added at the ITR.

[0092] Table 4. Verification results of virus production in the case of stable insertion of the transgene cassette into the genome (vg)

[0093]

[0094]

Claims

1. A polynucleotide, characterized in that, The polynucleotide comprises: an adeno-associated virus (AAV) 5' inverted terminal repeat (ITR) sequence, a recombinant adeno-associated virus (rAAV) transgene cassette sequence, an AAV 3' ITR sequence, and one or both of an insulator sequence or its reverse sequence linked to the 5' ITR sequence and an insulator sequence or its reverse sequence linked to the 3' ITR sequence.

2. The polynucleotide of claim 1, wherein, The insulator sequence or its reverse sequence linked to the 5' ITR sequence is linked to the 5' end of the 5' ITR sequence.

3. The polynucleotide of claim 1, wherein The insulator sequence or its reverse sequence linked to the 3' ITR sequence is linked to the 3' end of the 3' ITR sequence.

4. The polynucleotide of claim 1, wherein The insulator sequence is derived from the AAVS1 region of human chromosome 19.

5. The polynucleotide of claim 1, wherein The insulator sequence comprises or is derived from a DNase 1 hypersensitive site in the AAVS1 region of human chromosome 19.

6. The polynucleotide of claim 1, wherein The insulator is an HS4 insulator.

7. Vector, characterized in that, The vector comprises the polynucleotide according to any one of claims 1-6.

8. A cell, characterized in that, The cell comprises the polynucleotide according to any one of claims 1-6 or the vector according to claim 7.

9. Use of the polynucleotide according to any one of claims 1-6, the vector according to claim 7, or the cell according to claim 8 in the manufacture of a rAAV vector.

10. A method for preparing an rAAV vector, characterized in that, The method comprises transfecting a cell with the polynucleotide according to any one of claims 1-6 or the vector according to claim 7.

11. The method of claim 10, wherein, The polynucleotide according to any one of claims 1-6 or the vector according to claim 7 is transiently transfected into the cell.

12. The method of claim 10, wherein, The polynucleotide according to any one of claims 1-6 is integrated into the genome of the cell.

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