Gene delivery vector and use thereof

Abstract

A gene delivery vector system, and a use thereof in delivering an ultra-long gene. The ultra-long gene is split into two or more parts, which are respectively placed in upstream and downstream vectors, and site-specific recombination systems are respectively introduced into the upstream and downstream vectors, so as to ultimately obtain a dual or multiple delivery system LegoVec. Compared with conventional systems, the present system exhibits an approximately 2- to 5-fold increase in cellular-level fluorescence intensity and an approximately 6-fold increase in RT-qPCR expression in animals. The present system enables efficient multi-vector gene recombination and expression efficiency, and provides a simple, rapid, and efficient gene delivery tool.

Description

Gene delivery vector and application thereof

[0001] This application claims priority to the Chinese patent application No. 202410426177.1, filed on April 9, 2024, and entitled "Gene delivery vector and application thereof", the entire content of which is incorporated herein by reference. TECHNICAL FIELD

[0002] The present application relates to the field of biotechnology, and in particular to a gene delivery vector and its application in delivering an ultralong gene. BACKGROUND

[0003] Since the FDA approved the first gene therapy for ADA-SCID in 1990, after more than 30 years of twists and turns, gene therapy has entered a period of rapid development. Not only has its safety and effectiveness been widely recognized, but its indications have also expanded from early congenital genetic diseases to tumors, cardiovascular diseases, autoimmune diseases, and infectious diseases. Currently, gene therapy vectors are mainly divided into non-viral vectors and viral vectors. Compared with non-viral vectors, viral vectors have higher targeting and delivery efficiency, and therefore are the mainstream vectors in gene therapy, accounting for more than 60% in gene therapy.

[0004] In recombinant viral vectors that have been genetically engineered, although the sequences in the viral genome that are not related to viral packaging have been deleted to the maximum extent, the upper limit of the packaging capacity cannot exceed the length of the wild-type genome. Therefore, the space left for exogenous sequences is still limited. Taking AAV as an example, the loading capacity of AAV is small, and it can only accommodate a 4.7-kb DNA fragment including ITR. This greatly hinders the application of AAV in the treatment of genetic diseases with pathogenic genes longer than 4.7 kb, such as Stargardt disease (STGD) and Usher syndrome type IB (USH1B).

[0005] To overcome the limitation of small AAV loading capacity, researchers introduced a dual-AAV (Dual-AAV) vector system, that is, a large gene is divided into two halves, and placed on two AAV vectors respectively. After the two vectors are packaged into viruses respectively, the target cells are co-infected, and the two AAV genomes in the same cell undergo intermolecular recombination, thereby expressing a complete exogenous protein. The structures of the two vectors of the dual-AAV system are ITR-promoter-5'CDS-SD-HA-ITR (Figure 1) and ITR-HA-SA-3'CDS-polyA-ITR (Figure 2) respectively, wherein HA is the homologous arm region, SD is the splice donor site, and SA is the splice acceptor site. The vector system superimposes two recombination effects. The first is the easy recombination between different AAV virus ITRs to form a circular multimeric DNA. The second is the homologous recombination between the upstream and downstream vectors to form a DNA product of ITR-promoter-5'CDS-SD-HA-SA-3'CDS-polyA. The transcription products of the two kinds of recombination DNA in the system will undergo splicing between SD and SA to form a mature mRNA with the structure of 5'Cap-full CDS-polyA, and further translate into a complete target protein. The system is called Dual-R. Compared with the conventional AAV vector, the dual-AAV system can accommodate longer exogenous sequences, expanding the scope of application of target indications. However, the prerequisite for dual-AAV recombination is that the two viruses need to enter the same cell at the same time and randomly collide to recombine. Therefore, under the same dose, the expression level is much lower than that of the conventional AAV expression vector. In order to improve the expression level of the large gene target protein and obtain significant therapeutic effect, the method of increasing the dose of the two AAV viruses is often adopted, but high-dose viruses often produce high cytotoxicity, which is extremely unfavorable for gene therapy. SUMMARY

[0006] Therefore, the present application provides a more efficient gene delivery vector system and its application in delivering an ultralong gene.

[0007] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0008] The gene delivery vector LegoVec comprises: n upstream vectors containing split fragments of a target gene and 1 downstream vector containing split fragments of the target gene.

[0009] The target gene comprises, in sequence from 5' end to 3' end, a first split fragment, a second split fragment,..., an (n-1)th split fragment, an nth split fragment, and an (n+1)th split fragment.

[0010] (1) when n is equal to 1, the upstream vector comprises an expression frame U, the expression frame U comprising a promoter, a first split fragment, a splice donor site 1, a recombination site 1A connected in sequence;

[0011] the downstream vector comprises an expression frame D, the expression frame D comprising a recombination enzyme gene expression cassette, a recombination site 1B, a splice acceptor site 1, a second split fragment, a PolyA terminator connected in sequence;

[0012] (2) when n is equal to 2, the upstream vector comprises an upstream vector 1# and an upstream vector 2#; the upstream vector 1# comprises an expression frame V1#, the expression frame V1# comprising a promoter, a first split fragment, a splice donor site 1, a recombination site 1A connected in sequence;

[0013] the upstream vector 2# comprises an expression frame V2#, the expression frame V2# comprising a recombination site 1B, a splice acceptor site 1, a second split fragment, a splice donor site 2, a recombination site 2A connected in sequence;

[0014] the downstream vector comprises an expression frame D#, the expression frame D# comprising a recombination enzyme gene expression cassette, a recombination site 2B, a splice acceptor site 2, a third split fragment and a PolyA terminator connected in sequence;

[0015] (3) when n is an integer greater than or equal to 3, the upstream vector comprises: an upstream vector 1, an upstream vector 2, …, an upstream vector n-1 and an upstream vector n;

[0016] the upstream vector 1 comprises an expression frame V1, the expression frame V1 comprising a promoter, a first split fragment, a splice donor site 1, a recombination site 1A connected in sequence;

[0017] the upstream vector n-i comprises an expression frame Vn-i, the expression frame Vn-i comprising a recombination site (n-i-1)B, a splice acceptor site n-i-1, an n-i split fragment, a splice donor site n-i, a recombination site (n-i)A connected in sequence; 1≤i≤n-2, i is an integer;

[0018] the upstream vector n comprises an expression frame Vn, the expression frame Vn comprising a recombination site (n-1)B, a splice acceptor site n-1, an n split fragment, a splice donor site n, a recombination site nA connected in sequence;

[0019] the downstream vector comprises an expression frame D*, the expression frame D* comprising a recombination enzyme gene expression cassette, a recombination site nB, a splice acceptor site n, an n+1 split fragment and a PolyA terminator connected in sequence;

[0020] the recombination enzyme gene expression cassette comprises a promoter, a recombination enzyme gene and a PolyA terminator;

[0021] The recombination sites 1, 2,..., n are selected from different sites in the same recombination system, and nA and nB can undergo a recombination reaction.

[0022] In some embodiments, the gene delivery system of the present application, the recombination enzyme gene expression cassette is deleted by recombination, specifically, the gene delivery system comprises:

[0023] (1) when n is equal to 1, the upstream vector further comprises a recombination site 2A at either end of the recombination site 1A in the expression frame U of the upstream vector;

[0024] The downstream vector further comprises a recombination site 2B at either end of the recombination enzyme gene in the expression frame D of the downstream vector;

[0025] (2) when n is equal to 2, the upstream vector 2# further comprises a recombination site 3A at either end of the recombination site 2A in the expression frame V2# of the upstream vector 2#;

[0026] The downstream vector further comprises a recombination site 3B at either end of the recombination enzyme gene in the expression frame D# of the downstream vector;

[0027] (3) when n is an integer greater than or equal to 3, the upstream vector n further comprises a recombination site (n+1)A at either end of the recombination site nA in the expression frame Vn of the upstream vector n;

[0028] The downstream vector further comprises a recombination site (n+1)B at either end of the recombination enzyme gene in the expression frame D* of the downstream vector.

[0029] In other embodiments, the gene delivery system of the present application, the expression of the recombination enzyme is controlled by a binding protein splicing system, and the recombination enzyme gene expression cassette comprises an N-terminal recombination enzyme gene expression cassette and a C-terminal recombination enzyme gene expression cassette, and the two expression cassettes are located in any two of n upstream vectors and 1 downstream vector. Specifically, the gene delivery vector comprises:

[0030] n upstream vectors containing split fragments of the target gene and 1 downstream vector containing split fragments of the target gene;

[0031] The target gene comprises, from 5' to 3', a first split fragment, a second split fragment,..., an (n-1)th split fragment, an nth split fragment, and an (n+1)th split fragment connected in sequence;

[0032] (1) when n is equal to 1, the upstream vector comprises an expression frame U', which comprises, in sequence, a promoter, a first split fragment, a splicing donor site 1, and a recombination site 1A;

[0033] The downstream vector comprises an expression frame D', which comprises recombination site 1B, splice acceptor site 1, 2nd split fragment, PolyA terminator connected in sequence;

[0034] Any one end of the upstream vector expression frame U' further comprises an N-terminal or C-terminal recombinase gene expression cassette,

[0035] Any one end of the downstream vector expression frame D' further comprises a C-terminal or N-terminal recombinase gene expression cassette;

[0036] The N-terminal recombinase gene expression cassette and the C-terminal recombinase gene expression cassette are respectively located in any two vectors of the n upstream vectors and 1 downstream vector;

[0037] The N-terminal recombinase gene expression cassette comprises a promoter, an N-terminal recombinase gene fragment-protein splice donor site and a PolyA terminator; and the C-terminal recombinase gene expression cassette comprises a promoter, a protein splice acceptor site-C-terminal recombinase gene fragment and a PolyA terminator;

[0038] (2) When n is equal to 2, the upstream vector comprises upstream vector 1# and upstream vector 2#; the upstream vector 1# comprises an expression frame V1#', which comprises a promoter, a 1st split fragment, a splice donor site 1 and a recombination site 1A connected in sequence;

[0039] The upstream vector 2#' comprises an expression frame V2#', which comprises a recombination site 1B, a splice acceptor site 1, a 2nd split fragment, a splice donor site 2 and a recombination site 2A connected in sequence;

[0040] The downstream vector comprises an expression frame D#', which comprises a recombination site 2B, a splice acceptor site 2, a 3rd split fragment and a PolyA terminator connected in sequence;

[0041] Any one end of the expression frame of the upstream vector 1# or 2# further comprises an N-terminal or C-terminal recombinase gene expression cassette;

[0042] Any one end of the downstream vector expression frame D' further comprises a C-terminal or N-terminal recombinase gene expression cassette;

[0043] The N-terminal recombinase gene expression cassette and the C-terminal recombinase gene expression cassette are respectively located in any two vectors of the n upstream vectors and 1 downstream vector;

[0044] The N-terminal recombinase gene expression cassette comprises a promoter, an N-terminal recombinase gene fragment-protein splice donor site and a PolyA terminator; and the C-terminal recombinase gene expression cassette comprises a promoter, a protein splice acceptor site-C-terminal recombinase gene fragment and a PolyA terminator;

[0045] (3) n is an integer greater than or equal to 3, the upstream vectors include: upstream vector 1, upstream vector 2,..., upstream vector n-1 and upstream vector n;

[0046] The upstream vector 1 comprises an expression frame V1', which comprises a promoter, a first split fragment, a splice donor site 1, a recombination site 1A connected in sequence;

[0047] The upstream vector n-i' comprises an expression frame Vn-i', which comprises a recombination site (n-i-1)B, a splice acceptor site n-i-1, a n-i split fragment, a splice donor site n-i, a recombination site (n-i)A connected in sequence; 1≤i≤n-2, i is an integer;

[0048] The upstream vector n' comprises an expression frame Vn', which comprises a recombination site (n-1)B, a splice acceptor site n-1, a n split fragment, a splice donor site n, a recombination site nA connected in sequence;

[0049] The downstream vector comprises an expression frame D*', which comprises a recombination site nB, a splice acceptor site n, a n+1 split fragment and a PolyA terminator connected in sequence;

[0050] Any one end of the expression frame of the upstream vector 1-n further comprises an N-terminal or C-terminal recombinase gene expression cassette;

[0051] Any one end of the downstream vector expression frame D' further comprises a C-terminal or N-terminal recombinase gene expression cassette;

[0052] The N-terminal recombinase gene expression cassette and the C-terminal recombinase gene expression cassette are located in any two of the n upstream vectors and 1 downstream vector, respectively;

[0053] The N-terminal recombinase gene expression cassette comprises a promoter, an N-terminal recombinase gene fragment-protein splice donor site and a PolyA terminator; the C-terminal recombinase gene expression cassette comprises a promoter, a protein splice acceptor site-C-terminal recombinase gene fragment and a PolyA terminator;

[0054] The recombination sites 1, 2,..., n are selected from different sites in the same recombination system, and nA and nB can undergo a recombination reaction.

[0055] In some embodiments, the gene delivery vector comprises:

[0056] (1) n is equal to 1, and any one end of the recombination site 1A in the expression frame U' of the upstream vector is further connected with a recombination site 2A;

[0057] In the expression frame D' of the downstream vector, either end of the N-terminal recombinase gene fragment-protein splicing donor site or protein splicing acceptor site-C-terminal recombinase gene fragment is further connected with a recombination site 2B;

[0058] (2) When n is equal to 2, in the expression frame V2' of the upstream vector 2#, either end of the recombination site 2A is further connected with a recombination site 3A;

[0059] In the expression frame D' of the downstream vector, either end of the N-terminal recombinase gene fragment-protein splicing donor site or protein splicing acceptor site-C-terminal recombinase gene fragment is further connected with a recombination site 3B;

[0060] (3) When n is an integer greater than or equal to 3, in the expression frame Vn' of the upstream vector n, either end of the recombination site nA is further connected with a recombination site (n+1)A;

[0061] In the expression frame D' of the downstream vector, either end of the N-terminal recombinase gene fragment-protein splicing donor site or protein splicing acceptor site-C-terminal recombinase gene fragment is further connected with a recombination site (n+1)B.

[0062] In the above vectors, the mentioned recombination site n and recombination site n+1 are selected from any one of the following recombination systems: Cre / lox, Dre / rox, FLP / FRT, FLPo / FRT, Vika / vox, λ-Int / attP, B2 / B2RT, B3 / B3RT, R / R1RT, T / TDRT, Kw / KwRT, KD / KDRT, ΦC31 / att, γδ / att, Tn3 / att, Gin / att, Bxb1 / att or R4 / att; n is selected from an integer greater than or equal to 1.

[0063] In some embodiments, the lox site includes but is not limited to loxP, lox71, lox75, lox44, lox66, lox76, lox43, lox72, lox78, lox65, lox511, lox512, lox514, lox5171, lox2272, loxM2, loxM3, loxM7 and loxM11, m2, m3, m7 or m11 variants;

[0064] The rox site includes but is not limited to roxP, rox1, rox2, rox7, rox8, rox9, rox12, rox34, rox61 or rox85 variants;

[0065] The FRT site includes but is not limited to FRT, F1, F2, F3, F4 or F5 variants;

[0066] The att site includes, but is not limited to, attB, attP, attL or attR variants.

[0067] In the gene delivery system of the present application, the total length of the sequence after recombination of the upstream and downstream vectors is ≥ 4.7 kb.

[0068] The backbone of the gene delivery vector of the present application is selected from an AAV adenovirus-associated vector, an adenovirus vector, an HSV vector, a poxvirus vector, a baculovirus vector, a conventional gene expression vector, a transposon vector, a prokaryotic expression vector, a yeast expression vector, a zebrafish expression vector, a fruit fly expression vector, a plant expression vector or a nematode expression vector.

[0069] In the present application, the promoter includes a ubiquitous expression promoter, a tissue-specific expression promoter or an inducible expression promoter, and the present application does not have a special requirement for the specific type of the promoter, and any one selected from the above-mentioned promoters can be used, such as CMV, EF1A, EFS, CAG, CBh, SFFV, MSCV, SV40, mPGK, hPGK, UBC, RSV, Nanog, Nes, Tubala, Camk2a, SYN1, Hb9, Th, NSE, GFAP, Ibal, ProA1, hRK, hRHO, hBEST1, Prnp, Cnp, K14, BK5, mTyr, cTnT, aMHC, Myog, ACTA1, MHCK7, SM22a, EnSM22a, Runx2OC, Collal, Col2al, aP2, Adipoq, Tiel, Cd144, CD68, CD1b, Afp, Alb, TBG, MMTV, Wap, HIP, Pdxl, Ins2, Hcn4, NPHS2, SPB, CD144, TERT, TRE, TRE3G, GAL1, MET17, CUP1, AOX1, sCMV, bactin2, Ubi, cmlc2, zK5, 503unc, HSP70, 5x UAS, CaMV35S, Nos, ZmUbi, TEF1, GPD, ADH1, GAP, actin5C, Polyubiquitin, a 1-tubulin, Rh2, Mtn, U6, U3, H1, U6-26, TK, RSV, MC1, GAL1, PH, p5, p10, p40, p41, araBAD, cspA, Hsp68, pL, pR, EM7, T7, SP6, AmpR promoter, etc., including but not limited to the above. In some specific embodiments, the promoter is selected from CMV and CAG.

[0070] In the present application, the terminator is a commonly used terminator, such as a SV40 pA terminator, a rBGpA terminator, a BGHpA terminator, or a hGH terminator.

[0071] The recombinase can mediate efficient recombination of the recombination site to produce deletion, inversion, and translocation of specific fragments, and realize controllable expression of genes in vivo and in vitro. In the present application, the recombinase gene expression cassette can express the recombinase, which is used for recombination of split fragments of the target gene, and can be located in any one or several expression frames of the upstream vector and / or the downstream vector, i.e., at least one of the expression frames of the expression frame U, the expression frame V1#, the expression frame V2#, the expression frame V1, the expression frame Vn-i, the expression frame Vn, the expression frame D, the expression frame D, and the expression frame D* contains the recombinase gene expression cassette.

[0072] In some embodiments, at least one of the expression frame D, the expression frame D#, and / or the expression frame D* contains the recombinase gene expression cassette, which is located at the 3' end of the expression frame.

[0073] In other embodiments, any one of the expression frame U, the expression frame V1#, the expression frame V2#, the expression frame V1, the expression frame Vn-i, and the expression frame Vn further contains the recombinase gene expression cassette; the recombinase gene expression cassette is located at the 3' end and / or the 5' end of the expression frame.

[0074] In other embodiments, when the expression frame D, the expression frame D#, and the expression frame D* do not contain the recombinase gene expression cassette, the recombinase gene expression cassette is located at the 3' end and / or the 5' end of any one of the expression frame U, the expression frame V1#, the expression frame V2#, the expression frame V1, the expression frame Vn-i, or the expression frame Vn.

[0075] As long as the specific position of the recombinase gene expression cassette in the expression frame does not affect the cleavage of the cleavage site and the expression of the gene, it is feasible. Studies have shown that compared with other feasible positions, the recombinase gene expression cassette is located at any one end of the expression frame, which has higher cleavage efficiency, and is a preferred scheme of the present application.

[0076] Specifically, in some preferred schemes of the present application, the recombinase gene expression cassette is located in the downstream vector, preferably in the expression frame of the downstream vector. Specifically, it is located at any one end of the expression frame D, the expression frame D#, or the expression frame D*, i.e., the 5' end or the 3' end of the expression frame. In a specific embodiment of the present application, the recombinase gene expression cassette is located at the 5' end of the recombination site or the 3' end of the PolyA terminator.

[0077] In some preferred embodiments of the present application, the recombinase gene expression cassette is located in the upstream vector, preferably in the expression frame of the upstream vector. It can be located at either end of the expression frame U, expression frame V1#, expression frame V2#, expression frame V1, expression frame Vn-i or expression frame Vn, including the 3' end and 5' end of the expression frame. Taking expression frame U as an example, the expression frame U comprises a promoter, a first split fragment, a splice donor site 1, and a recombination site 1A connected in sequence. The recombinase gene expression cassette is located at either end of the expression frame U, i.e. the 5' end or the 3' end, which means that the recombinase gene expression cassette is located at the 5' end of the promoter or the 3' end of the recombination site 1A.

[0078] The present application also provides a virus packaged by a packaging cell using the gene delivery vector described above.

[0079] The packaging cell includes HEK293, HEK293T, HEK293A, HeLa, BHK21, Sf9 and / or CHO.

[0080] The gene delivery vector described in the present application or the gene delivery vector prepared by the preparation method described in the present application is used in the preparation of the virus described in the present application, and is used in the preparation of a gene delivery drug or in the delivery of an ultralong gene.

[0081] The present application also provides a gene delivery drug comprising the gene delivery vector described in the present application. BRIEF DESCRIPTION OF DRAWINGS

[0082] Figure 1 shows the vector structure of ITR-promoter-5'CDS-SD-HA-ITR;

[0083] Figure 2 shows the vector structure of ITR-HA-SA-3'CDS-polyA-ITR;

[0084] Figure 3 shows the schematic diagram of the upstream vector structure of

promoter 1-5'CDS-SD-lox1-lox2

[0085] Figure 4 shows the schematic diagram of the downstream vector structure of

promoter 2-lox2-InCre-PolyA1-lox1-SA-3'CDS-PolyA2

[0086] Figure 5 shows the schematic diagram of the vector structure of

promoter 1-5'CDS-SD-lox1-SA-3'CDS-polyA2

[0087] Figure 6 shows the map of the vector pAAV[Exp]-CAG>5'EGFP-SD-AK;

[0088] Figure 7 shows a map of vector pAAV[Exp]-AK-SA-3'EGFP-SV40 late pA;

[0089] Figure 8 shows a map of vector pAAV[Exp]-CAG>5'EGFP-SD-loxP-lox2272;

[0090] Figure 9 shows a map of vector pAAV[Exp]-CMV>lox2272-InCre-SV40 late pA-loxP-SA-3'EGFP-BGH pA;

[0091] Figure 10 shows a map of vector pAAV[Exp]-CAG>EGFP:WPRE;

[0092] Figure 11 shows a comparison of fluorescence intensity of Dual-lox system and Dual-R system at cell level;

[0093] Figure 12 shows a comparison of relative expression of EGFP in Dual-lox and Dual-R system at animal level;

[0094] Figure 13 shows a map of vector pAAV[Exp]-CAG>5'EGFP-SD-roxP-rox12;

[0095] Figure 14 shows a map of vector pAAV[Exp]-CMV>rox12-InDre-SV40 late pA-roxP-SA-3'EGFP-BGH pA;

[0096] Figure 15 shows a map of vector pAAV[Exp]-CAG>5'EGFP-SD-FRT-F5;

[0097] Figure 16 shows a map of vector pAAV[Exp]-CMV>F5-InFlpo-SV40 late pA-FRT-SA-3'EGFP

[0098] -BGH pA;

[0099] Figure 17 shows a flow cytometry analysis of fluorescence rate of Dual-rox, Dual-FRT and Dual-lox;

[0100] Figure 18 shows a flow cytometry analysis of fluorescence intensity of Dual-rox, Dual-FRT and Dual-lox;

[0101] Figure 19 shows a map of vector pAAV[Exp]-CAG>5'EGFP-SD-loxP-lox2272-81pA-CMV>5'Cre(aa248-CRV)-InN-81pA;

[0102] Figure 20 shows the vector map of pAAV[Exp]-CMV>lox2272-InC-3'Cre(aa248-CRV)-81pA-loxP-SA-3'EGFP-81pA

[0103] Figure 20 shows the vector map of pAAV[Exp]-CMV>lox2272-InC-3'Cre(aa248-CRV)-81pA-loxP-SA-3'EGFP-81pA

[0104] Figure 21 shows the fluorescence rate of Dual-lox and Dual-loxI detected by flow cytometry analysis;

[0105] Figure 22 shows the fluorescence intensity of Dual-lox and Dual-loxI detected by flow cytometry analysis;

[0106] Figure 23 shows the vector map of pRP[Exp]ΔpA-Kan-CAG>dys(1-3945)-SD-loxP

[0107] Figure 24 shows the vector map of pRP[Exp]ΔpA-Kan-CMV>Incre-polyA-loxP-SA-dys(3946-6631)-SD-lox2272

[0108] Figure 25 shows the vector map of pRP[Exp]-Lox2272-SA-dys(6632-11055)-poyA

[0109] Figure 26 shows the WB detection results of DMD protein in Example 5. DETAILED DESCRIPTION

[0110] The present application provides a gene delivery vector and its application in delivering super-long genes. Those skilled in the art can refer to the content herein and make appropriate improvements to the process parameters. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The methods and applications of the present application have been described by preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the methods and applications herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0111] The test materials used in the present application are all ordinary commercially available products and can be purchased in the market.

[0112] The gene delivery vector provided by the application ingeniously introduces a site-specific recombination system, such as Cre / lox (lox sites include loxP, lox2272, lox71, lox66, lox511, lox5171, m2, m3, m7, m11, etc.), Dre / rox (rox sites include roxP, rox1, rox2, rox7, rox8, rox9, rox12, rox34, rox61, rox85, etc.), FLP (or codon-optimized FLPo) / FRT (FRT sites include FRT, F1, F2, F3, F4, F5, etc.), PhiC31 / att (att sites include attB, attP, attL, attR, etc.), B2 / B2RT, B3 / B3RT, R / R1RT, T / TDRT, KD / KDRT, etc., to obtain a more efficient double or multi-vector delivery system tool, named LegoVec, which can more efficiently deliver genes. The structure is shown in the following examples. Upstream vector 1: promoter 1-5'CDS-SD-recombination site 1A-recombination site 2A; downstream vector 2: promoter 2-recombination site 2B-site recombination enzyme-PolyA terminator 1-recombination site 1B-SA-3'CDS-PolyA terminator 2. The downstream vector expresses the site recombination enzyme, so that the recombination sites of the upstream and downstream vectors are subjected to intermolecular recombination. If recombination site 1 recombines first, the product formed is promoter 1-5'CDS-SD-recombination site 1-SA-3'CDS-PolyA terminator 2. The mRNA of the recombination product can be cleaved through the SD and SA sites, and then a complete large gene 5'Cap-full CDS-PolyA terminator 2 capable of expression is formed. If recombination site 2 recombines first, the product formed is promoter 1-5'CDS-SD-recombination site 1-recombination site 2-recombination enzyme-PolyA terminator 1-recombination site 1-SA-3'CDS-PolyA terminator 2. Recombination site 1 further undergoes intramolecular recombination, and then promoter 1-5'CDS-SD-recombination site 1-SA-3'CDS-PolyA terminator 2 is formed. The mRNA of the recombination product can be cleaved through the SD and SA sites, and then a complete large gene 5'Cap-full CDS-PolyA terminator 2 capable of expression is formed. After recombination, the promoter 2 is deleted, so that the recombination enzyme cannot be further expressed, thereby avoiding the toxicity reaction caused by excessive expression of the recombination enzyme. Alternatively, recombination site 2 can be removed according to the application requirements, so that the recombination enzyme is expressed for a long time or is specifically or inducibly expressed. Alternatively, the recombination enzyme can be divided into two parts of N-terminal and C-terminal, and is fused with Intein protein cleavage system InN and InC, respectively, and is arranged in the upstream and downstream vectors, so as to combine the protein cleavage system to control the expression of the recombination enzyme.If a larger gene needs to be delivered, the larger gene can be truncated into more upstream vectors, and by introducing more recombination site variants or another set of recombination systems, upstream vector 2, upstream vector 3, etc. to achieve the purpose of expansion. Taking the Cre / lox system as an example, the system is a site-specific recombination system derived from P1 phage, and the Cre recombinase can mediate the efficient recombination of the lox site to produce specific fragment deletion, inversion, translocation, and realize the regulated expression of genes in vivo and in vitro. The recombination reaction is reversible or irreversible, and researchers have derived various lox mutants such as lox2272, lox71, lox66, lox511, lox5171, m2, m3, m7, m11, etc. by mutating the wild-type loxP site. These lox mutant sites can also be recognized by Cre enzyme. Using this property, we introduced lox1 and lox2 sites with different spacer sequences on the upstream and downstream vectors, based on AAV viral vectors, the upstream vector structure is

Promoter1-5’CDS-SD-lox1-lox2

Promoter2-lox2-InCre-PolyA1-lox1-SA-3’CDS-PolyA2

Promoter1-5’CDS-SD-lox1-SA-3’CDS-polyA2

[0113] The present application is further described below in conjunction with examples:

[0114] Example 1 Comparison of Dual-lox and Dual-R systems at the cellular level

[0115] 1. Based on Cre / Lox recombination system, the following five vectors are constructed:

[0116] Vector 1: pAAV[Exp]-CAG>5'EGFP-SD-AK

[0117] The vector map is shown in Figure 6.

[0118] 1.1 Construction of pDown-5'EGFP-SD-AK

[0119] Using pDown-EGFP as a template, the 400bp Esp3I-5'EGFP-Esp3I fragment is amplified by primers Esp3I-5'EGFP-F and Esp3I-5'EGFP-R and gel recovered, and then GoldenGate reaction is performed with the backbone pDown-Esp3I-ccdB-Cm-Esp3I-SD-AK. After transformation, single colonies are picked for PCR identification, and the correct pDown-5'EGFP-SD-AK clone is verified by enzyme digestion and sequencing. The primer sequences are as follows:

[0120] Esp3I-5'EGFP-F:

[0121] Esp3I-5'EGFP-R:

[0122] 1.2 Construction of pAAV[Exp]-CAG>5'EGFP-SD-AK

[0123] LR reaction is performed with the gateway clones pUp-CAG and pDown-5'EGFP-SD-AK and the backbone vector pAAV.Des2d(L-AEX / R-PAA). After transformation, single colonies are picked for PCR identification, and the correct pAAV[Exp]-CAG>5'EGFP-SD-AK clone is verified by enzyme digestion and sequencing.

[0124] Vector 2: pAAV[Exp]-AK-SA-3'EGFP-SV40 latepA

[0125] The vector map is shown in Figure 7.

[0126] Bbs I enzyme pAAV-Asc I-Eco RV-Xho I-Bbs I-ccdB-Cm-Bbs I-Pac I-Age I-Avr II, 3.5 kb of the skeleton fragment was recovered, and the AK-SA-3'EGFP-SV40 late pA fragment of 764 bp was amplified using the primer AK-F and 3'EGFP-SV40 late pA-R as a template and gel recovery, and the skeleton fragment recovered after enzyme digestion was subjected to infusion reaction, and the correct pAAV[Exp]-AK-SA-3'EGFP-SV40 late pA clone was identified by PCR after transformation, and enzyme digestion and sequencing verification. The primer sequences are as follows:

[0127] AK-F:

[0128] 3'EGFP-SV40 late pA-R:

[0129] Vector 3: pAAV[Exp]-CAG>5'EGFP-SD-loxP-lox2272

[0130] The vector map is shown in Figure 8.

[0131] 3.1 The 5'EGFP-SD-loxP-lox2272 fragment was synthesized by the gene synthesis company and cloned into the pDONR221 vector to form the entry clone pDown-5'EGFP-SD-loxP-lox2272.

[0132] 3.2 The entry clones pUp-CAG and pDown-5'EGFP-SD-loxP-lox2272 were used to perform LR reaction with the skeleton vector pAAV.Des2d(L-AEX / R-PAA), and the correct pAAV[Exp]-CAG>5'EGFP-SD-loxP-lox2272 clone was identified by PCR after transformation, and enzyme digestion and sequencing verification.

[0133] Vector 4: pAAV[Exp]-CMV>lox2272-InCre-SV40 late pA-loxP-SA-3'EGFP-BGHpA, and the vector map is shown in Figure 9.

[0134] 4.1 Construction of pDown-lox2272-InCre-SV40 late pA-loxP-SA-3'EGFP

[0135] Synthesis of fragment 1 : lox2272-InCre; InCre-SV40 late pA fragment was amplified by PCR using primers Go-221002-1003bfb-PF2 and Go-221002-1003bfb-PR2 (63.7) with pUp-TRE:InCre:SV40 late pA as template; SA-3'EGFP was amplified by PCR using primers Go- 221002-1004ded-PF3 (64.6) and Go-221002-1003bfb-PR3 (65.2) with pDown-AK-SA-3'EGFP-SV40 late pA as template. The three fragments above were used for GoldenGate reaction with backbone vector pDown-BsaI-ccdB-Cm-BsaI, and the correct pDown-lox2272-InCre-SV40 late pA-loxP-SA-3'EGFP clone was identified by PCR, and verified by enzyme digestion and sequencing. Primer sequences are as follows:

[0136] Go-221002-1003bfb-PF2 (64.6):

[0137] Go-221002-1003bfb-PR2 (63.7)

[0138] Go-221002-1004ded-PF3

[0139] Go-221002-1003bfb-PR3 (65.2)

[0140] 4.2 Construction of pAAV[Exp]-CMV>lox2272-InCre-SV40 late pA-loxP-SA-3'EGFP-BGHpA

[0141] LR reaction was performed with pUp-CMV and pDown-lox2272-InCre-SV40 late pA-loxP-SA-3'EGFP and backbone vector pAAV.Des2d(px601-5'ITR modified), and the correct pAAV[Exp]-CMV>lox2272-InCre-SV40 late pA-loxP-SA-3'EGFP-BGHpA clone was identified by PCR, and verified by enzyme digestion and sequencing.

[0142] Vector 5: pAAV[Exp]-CAG>EGFP:WPRE, the vector structure is shown in Figure 10.

[0143] LR reaction was performed with the entry clones pUp-CAG and pDown-EGFP and the backbone vector pAAV.Des2d, and after transformation, single clones were picked for PCR identification, and the correct pAAV[Exp]-CAG>EGFP:WPRE was verified by enzyme digestion and sequencing.

[0144] 1. The above 5 vectors were packaged into AAV2 virus to obtain super-purified AAV viruses 1-5:

[0145] 2. The obtained viruses were subjected to HEK293T cell transduction experiment

[0146] 3.1 One day before transduction, 293T cells were inoculated on a 12-well plate at a quantity of 3E+6 cells / 12-well plate;

[0147] 3.2 Viruses combination 1+2, 3+4, 5 were respectively transduced into HEK293T cells at a single virus dose of 1:1 with MOI=1E+4, wherein combination 1+2 is based on the Dual-R system, combination 3+4 is based on the Dual-lox system of the application, and virus 5 is a positive control.

[0148] 3.3 After 15h, the virus mixture was discarded and replaced with fresh DMEM complete medium;

[0149] 3.4 Flow analysis was performed 72h later to detect fluorescence efficiency and fluorescence intensity.

[0150] Result analysis: the average fluorescence intensity of the Dual-R system is 36753, while the average fluorescence intensity of the Dual-lox system is 72945. Compared with the Dual-R system, the fluorescence intensity is increased by about 2 times, and the cell level Dual-lox system has higher recombination efficiency than the Dual-R system, and the related analysis is shown in Figure 11.

[0151] Example 2: Comparison of Dual-lox and Dual-R systems at animal level

[0152] 1. The above 5 vectors were packaged into AAV2 virus to obtain super-purified AAV viruses 1-5:

[0153] Vector 1: pAAV[Exp]-CAG>5’EGFP-SD-AK, the map is shown in Figure 6.

[0154] Vector 2: pAAV[Exp]-AK-SA-3’EGFP-SV40 latepA, the map is shown in Figure 7.

[0155] Vector 3: pAAV[Exp]-CAG>5'EGFP-SD-loxP-lox2272, the map is shown in Figure 8.

[0156] Vector 4: pAAV[Exp]-CMV>lox2272-InCre-SV40 late pA-loxP-SA-3'EGFP-BGHpA, the map is shown in Figure 9.

[0157] 2. Virus combinations 1+2 (Dual-R), 3+4 (Dual-lox) were mixed in a 1:1 single virus dose manner to a concentration of 2E+8 / μl and 2E+7 / μl, respectively.

[0158] 3. 1 μl of each of the above two virus mixtures was taken for subretinal injection in mice, and obvious retinal elevation was observed.

[0159] 4. One month later, the eyeballs were taken, and the optic cups were taken for RNA extraction and reverse transcription into cDNA.

[0160] 5. RT-qPCR was used to detect the expression level of EGFP under different injection doses.

[0161] Results analysis: According to the relative quantitative RT-qPCR, the expression level of EGFP was increased by about 6 times in Dual-lox compared with Dual-R, whether in the dose of 1E+8 or 1E+7. The Dual-lox system had higher recombination efficiency than the Dual-R system in vivo animal level. The specific statistical analysis is shown in Figure 12.

[0162] Example 3 Test of delivery tools based on multiple recombination systems

[0163] 1. Based on the Dre / rox recombination system, the following vectors were constructed:

[0164] Vector 1: pAAV[Exp]-CAG>5'EGFP-SD-roxP-rox12, the vector structure is shown in Figure 13.

[0165] 1.1 Construction of pDown-5'EGFP-SD-roxP-rox12

[0166] Using pDown-EGFP as a template, 5'EGFP fragment was amplified by PCR using primers Go-230731-1869njw-PF1 and Go-230731-1869njw-PR1;

[0167] Synthesis of SD-roxP-rox12 fragment, GoldenGate reaction of two fragments with backbone pDown-Esp3I-ccdB-Cm-Esp3I, after transformation, single clone was picked for PCR identification, and the correct pDown-5'EGFP-SD-roxP-rox12 clone was verified by enzyme digestion and sequencing. The primer sequences are as follows:

[0168] Go-230731-1869njw-PF1 (65.3):

[0169] Go-230731-1869njw-PR1 (64.2):

[0170] 1.2 Construction of pAAV[Exp]-CAG>5'EGFP-SD-roxP-rox12

[0171] LR reaction of the entry clones pUp-CAG and pDown-5'EGFP-SD-roxP-rox12 with the backbone vector pAAV.Des2d(L-AEX / R-PAA) was performed, and after transformation, single clone was picked for PCR identification, and the correct pAAV[Exp]-CAG>5'EGFP-SD-roxP-rox12 clone was verified by enzyme digestion and sequencing.

[0172] Vector 2: pAAV[Exp]-CMV>rox12-InDre-SV40 late pA-roxP-SA-3'EGFP-BGHpA, the map is shown in Figure 14.

[0173] 1.3 Construction of pDown-rox12-InDre-SV40 late pA-roxP-SA-3'EGFP

[0174] PCR amplify InDre fragment using primers Go-230731-1873zex-PF1 (65.2) and Go-230731-1873zex-PR1 (64.6) with pDown-SDN as template; PCR amplify SV40 late pA fragment using primers Go-230731-1873zex-PF2 (65.1) and Go-230731-1873zex-PR2 (63.7) with pUC19(mini).Des2d.C / EGFP(G to A) as template; PCR amplify SA-3'EGFP fragment using primers Go-230731-1873zex-PF3 (66.2) and Go-230731-1873zex-PR3 (65.2) with pDown-5'EGFP-SD-SA-3'EGFP as template; perform GoldenGate reaction with three fragments and backbone pDown-BsaI-ccdB-Cm-BsaI, pick single clone after transformation, and perform enzyme digestion and sequencing to verify correct pDown-rox12-InDre-SV40 late pA-roxP-SA-3'EGFP clone. Primer sequences are as follows:

[0175] Go-230731-1873zex-PF1 (65.2):

[0176] Go-230731-1873zex-PR1 (64.6):

[0177] Go-230731-1873zex-PF2 (65.1):

[0178] Go-230731-1873zex-PR2 (63.7):

[0179] Go-230731-1873zex-PF3 (66.2):

[0180] Go-230731-1873zex-PR3 (65.2):

[0181] 1.4 Construction of pAAV[Exp]-CMV>rox12-InDre-SV40 late pA-roxP-SA-3'EGFP-BGHpA

[0182] LR reaction was performed with the entry clone pUp-CMV and pDown-rox12-InDre-SV40 late pA-roxP-SA-3'EGFP and the backbone vector pAAV.Des2d(px601-5'ITR modified), after transformation, single clone was picked for PCR identification, and the correct pAAV[Exp]-CMV>rox12-InDre-SV40 late pA-roxP-SA-3'EGFP-BGH pA was verified by enzyme digestion and sequencing.

[0183] 2. Based on the Flp / FRT recombination system, the following vectors were constructed:

[0184] Vector 3: pAAV[Exp]-CAG>5'EGFP-SD-FRT-F5, the map is shown in Figure 15.

[0185] 3.1 Construction of pDown-5'EGFP:SD:FRT:F5

[0186] The 5'EGFP:SD:FRT:F5 fragment was directly synthesized and cloned into the backbone vector to form pDown-5'EGFP:SD:FRT:F5.

[0187] 3.2 Construction of pAAV[Exp]-CAG>5'EGFP-SD-FRT-F5

[0188] LR reaction was performed with the entry clone pUp-CAG and pDown-5'EGFP:SD:FRT:F5 and the backbone vector pAAV.Des2d(L-AEX / R-PAA), after transformation, single clone was picked for PCR identification, and the correct pAAV[Exp]-CAG>5'EGFP-SD-FRT-F5 was verified by enzyme digestion and sequencing.

[0189] Vector 4: pAAV[Exp]-CMV>F5-InFlpo-SV40 late pA-FRT-SA-3'EGFP-BGH pA, the map is shown in Figure 16.

[0190] 4.1 Construction of pDown-F5-InFlpo-SV40 late pA-FRT-SA-3'EGFP

[0191] InFlpo fragment was amplified from pDown-ORF_1365bp using primers Go-230808-1232qcb-PF1 (66.9) and Go-230808-1232qcb-PR1 (64.6); SV40 late pA-FRT fragment was amplified from pDown-dTom~T7 using primers Go-230808-1232qcb-PF2 (65.1) and Go-230808-1232qcb-PR2 (64.4); 3'EGFP fragment was amplified from pDown-EGFP using primers Go-230808-1232qcb-PF3 (65.8) and Go-230808-1232qcb-PR3 (62.9); the three fragments were subjected to GoldenGate reaction with backbone pDown-Esp3I-ccdB-Cm-Esp3I, and the correct pDown-F5-InFlpo-SV40 late pA-FRT-SA-3'EGFP clone was identified by PCR, enzyme digestion and sequencing. The primer sequences are as follows:

[0192] Go-230808-1232qcb-PF1 (66.9):

[0193] Go-230808-1232qcb-PR1 (64.6):

[0194] Go-230808-1232qcb-PF2 (65.1):

[0195] Go-230808-1232qcb-PR2 (64.4):

[0196] Go-230808-1232qcb-PF3 (65.8):

[0197] Go-230808-1232qcb-PR3 (62.9):

[0198] 4.2 Construction of pAAV[Exp]-CMV>F5-InFlpo-SV40 late pA-FRT-SA-3'EGFP-BGHpA

[0199] LR reaction was performed with the entry clone pUp-CMV and pDown-F5-InFlpo-SV40 late pA-FRT-SA-3'EGFP and the backbone vector pAAV.Des2d(px601-5'ITR modified), the transformants were picked and identified by PCR, and the correct pAAV[Exp]-CMV>F5-InFlpo-SV40 late pA-FRT-SA-3'EGFP-BGH pA was verified by enzyme digestion and sequencing.

[0200] 3. One day before transfection, 293T cells were seeded in 12-well plates at a density of 2.4E+6 cells / plate.

[0201] 4. On the day of transfection, the plasmids of group 1+2 and group 3+4 were diluted with base medium, and were added into the 12-well plates at a density of 2E+5 copies / cell, respectively, and the Dual-lox system was used as a parallel control.

[0202] 5. After 15h, the mixed solution was discarded and replaced with fresh DMEM complete medium.

[0203] 6. After 72h, flow cytometry was used to analyze the fluorescence efficiency and total fluorescence intensity, and the results are shown in Figures 17-18.

[0204] Result analysis: According to the results of Figures 17 and 18, both group 1+2 (Dual-rox) based on the Dre / rox recombination system and group 3+4 (Dual-FRT) based on the Flp / FRT recombination system can undergo efficient recombination, and the fluorescence rates of both are slightly higher than that of the Dual-lox system, and the fluorescence intensity is about 2.5 times that of the Dual-lox system.

[0205] Example 4. Controlling the expression of recombinase by using a protein splicing system to compare the efficiency of the Dual-lox and Dual-loxI systems at the cellular level

[0206] The Cre enzyme was divided into N-terminal and C-terminal two parts, and was fused with the protein splicing donor site InN and the protein splicing acceptor site InC, respectively, and was expressed on different vectors (referred to as Dual-loxI) to verify the recombination efficiency.

[0207] Vector 1:

[0208] pAAV[Exp]-CAG>5'EGFP-SD-loxP-lox2272-81pA-CMV>5'Cre(aa248-CRV)-InN-81pA, and the map is shown in Figure 19.

[0209] Construction process:

[0210] 1. Constructing intermediate vector 1:

[0211] pDown-5'EGFP-SD-loxP-lox2272-81pA-CMV>5'Cre(aa248-CRV)-InN-81pA

[0212] Synthetic fragment 5'EGFP-SD-loxP-lox2272; the second 589bp CMV fragment was amplified by PCR using primers Go-231010-1048twu-PF2 (63.9) and Go-231010-1048twu-PR2 (65.2) with pUp-CMV as template and gel recovery; the third 714bp 5'Cre(aa248-CRV) N-terminal fragment was amplified by PCR using primers Go-231010-1048twu-PF3 (68.5) and Go-231010-1048twu-PR3 (64.2) with pDown-Cre as template; the fourth fragment CRV)-InN-81pA was synthesized; the four fragments were subjected to Golden Gate reaction with the backbone pDown-Esp3I-ccdB-Cm-Esp3I, and the transformed single colonies were identified by PCR, and the correct pDown-5'EGFP-SD-loxP-lox2272-81pA-CMV>5'Cre(aa248-CRV)-InN-81pA clone was verified by enzyme digestion and sequencing. The primer sequences are as follows:

[0213] Go-231010-1048twu-PF2 (63.9):

[0214] Go-231010-1048twu-PR2 (65.2):

[0215] 2. Constructing final vector 1:

[0216] pAAV[Exp]-CAG>5'EGFP-SD-loxP-lox2272-81pA-CMV>5'Cre(aa248-CRV)-InN-81pA

[0217] The LR reaction was carried out by using the entry clone pUp-CAG and pDown-5'EGFP-SD-loxP-lox2272-81pA-CMV>5'Cre(aa248-CRV)-InN-81pA and the backbone vector pAAV.Des2d(L-AEX / R-PAA), and after transformation, single clones were picked for PCR identification, and the correct pAAV[Exp]-CAG>5'EGFP-SD-loxP-lox2272-81pA-CMV>5'Cre(aa248-CRV)-InN-81pA clone was verified by enzyme digestion and sequencing.

[0218] Vector 2:

[0219] pAAV[Exp]-CMV>lox2272-InC-3'Cre(aa248-CRV)-81pA-loxP-SA-3'EGFP-81pA, and the map is shown in Figure 20.

[0220] 3, Construction of intermediate vector 2:

[0221] pDown-lox2272-InC-3'Cre(aa248-CRV)-81pA-loxP-SA-3'EGFP-81pA

[0222] VB230817-1753may as a template, using primers

[0223] Go-231010-1050wxt-PF1(65.8) and Go-231010-1050wxt-PR1(64.2) PCR amplification of the first 460bp 3'Cre(aa248-CRV) C-terminal fragment and gel recovery; synthesis of the second 81pA-loxP-SA fragment; VB230920-1649kxm as a template, using primers Go-231010-1050wxt-PF3(65.8) and Go-231010-1050wxt-PR3(65.1) PCR amplification of the third 369bp 3'EGFP-81pA fragment; the above three fragments and the backbone pDown-Esp3I-ccdB-Cm-Esp3I were subjected to GoldenGate reaction, and after transformation, single clones were picked for PCR identification, and the correct pDown-lox2272-InC-3'Cre(aa248-CRV)-81pA-loxP-SA-3'EGFP-81pA clone was verified by enzyme digestion and sequencing.

[0224] The primer sequences are as follows:

[0225] Go-231010-1050wxt-PF1(65.8):

[0226] Go-231010-1050wxt-PR1(64.2):

[0227] Go-231010-1050wxt-PF3(65.8)

[0228] Go-231010-1050wxt-PR3(65.1)

[0229] 4. Construction of final vector 2: pAAV[Exp]-CMV>lox2272-InC-3'Cre(aa248-CRV)-81pA-loxP-SA-3'EGFP-81pA

[0230] Using the entry clone pUp-CMV and

[0231] pDown-lox2272-InC-3'Cre(aa248-CRV)-81pA-loxP-SA-3'EGFP-81pA was subjected to LR reaction with backbone vector pAAV.Des2d(L-AEX / R-PAA). After transformation, single clones were selected for PCR identification, and enzyme digestion and sequencing were performed to verify the correct pAAV[Exp]-CMV>

[0232] lox2272-InC-3'Cre(aa248-CRV)-81pA-loxP-SA-3'EGFP-81pA clone.

[0233] 5. Cell level testing

[0234] 5.1 One day before transfection, seed 293T cells in 12-well plates at 2.4E+6 cells / plate;

[0235] On the day of transfection, dilute the vector 1 + 2 (Dual-loxI) plasmids in basal medium and add 2E + 5 copies / cell to a 12-well plate. Use the Dual-lox system as a parallel control.

[0236] 5.3 After 15 hours, discard the mixture and replace with fresh DMEM complete medium;

[0237] 5.4 After 72 hours, flow cytometry was performed to detect the fluorescence efficiency and total fluorescence intensity. The results are shown in Figures 21 and 22.

[0238] Result analysis: According to the results of Fig. 21 and Fig. 22, both Dual-loxI and Dual-lox systems based on Cre / lox recombination system can occur high-efficiency recombination, and the fluorescence rates of the two are not much different, but in the fluorescence intensity, Dual-loxI is about 1.4 times higher than Dual-lox system.

[0239] Example 5 splitting and recombination of larger genes using the present application

[0240] The DMD gene is selected as the research object, the full-length DMD gene is 11 kb, the gene is split into three fragments, and the corresponding upstream vector and downstream vector are constructed.

[0241] 1. Construct the upstream vector 1 (Fig. 23): pRP[Exp]ΔpA-Kan-CAG>dys(1-3945)-SD-loxP, the upstream vector 1 contains the first split fragment 3945bp; construct the upstream vector 2 (Fig. 24): pRP[Exp]ΔpA-Kan-CMV>Incre-polyA-loxP-SA-dys(3946-6631)-SD-lox2272, the upstream vector 2 contains the second split fragment 2686bp and the recombinase gene expression box; construct the downstream vector (Fig. 25):

[0242] pRP[Exp]-Lox2272-SA-dys(6632-11055)-poyA, the downstream vector contains the third split fragment 4424bp;

[0243] 2. Equally transfect 6-well plate 293T cells with the three vectors, collect the cells after 48h and perform WB detection of DMD protein (antibody information: 12715-1-AP, proteintech).

[0244] Result analysis: As shown in the WB of Fig. 26, the signal of about 430kDa DMD full-length protein can be detected, indicating that the present application can realize the splitting and complete recombination of more gene fragments.

[0245] As shown by the results of Examples 1, 2, 3, 4 and 5, the present application introduces a site-specific recombination system to obtain a more efficient double or multi-vector delivery tool, which has a higher recombination efficiency than Dual-R system. The present application simply, quickly and efficiently realizes the balance between the dose and efficacy of the gene indication, and provides a more efficient gene delivery tool.

[0246] The above are only preferred embodiments of the present application, and it should be pointed out that for ordinary skilled persons in the art, some improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A gene delivery vector, characterized in that include: n upstream vectors containing target gene split fragments and 1 downstream vector containing target gene split fragments; The target gene includes a first split fragment, a second split fragment, ..., an n-1 split fragment, an nth split fragment and an n+1th split fragment connected sequentially from the 5' end to the 3' end; (1) When n is equal to 1, the upstream vector comprises an expression cassette U, which includes a promoter, a first split fragment, a splice donor site 1, and a recombination site 1A connected in sequence; The downstream vector comprises an expression cassette D, which includes a recombinase gene expression cassette, a recombination site 1B, a splice acceptor site 1, a second split fragment, and a PolyA terminator connected in sequence; (2) When n is equal to 2, the upstream vector includes upstream vector 1# and upstream vector 2#; the upstream vector 1# contains expression cassette V1#, and the expression cassette V1# includes a promoter, a first split fragment, a splice donor site 1, and a recombination site 1A connected in sequence; The upstream vector 2# comprises an expression cassette V2#, wherein the expression cassette V2# comprises a recombination site 1B, a splice acceptor site 1, a second split fragment, a splice donor site 2, and a recombination site 2A connected in sequence; The downstream vector comprises an expression cassette D#, wherein the expression cassette D# comprises a recombinase gene expression cassette, a recombination site 2B, a splice acceptor site 2, a third split fragment and a PolyA terminator connected in sequence; (3) When n is an integer ≥ 3, the upstream vectors include: upstream vector 1, upstream vector 2, ..., upstream vector n-1 and upstream vector n; The upstream vector 1 comprises an expression cassette V1, which includes a promoter, a first split fragment, a splice donor site 1, and a recombination site 1A connected in sequence; The upstream vector ni comprises an expression cassette Vn-i, wherein the expression cassette Vn-i comprises sequentially connected: a recombination site (ni-1) B, a splice acceptor site ni-1, the ni-th split fragment, a splice donor site ni, and a recombination site (ni) A; 1≤i≤n-2, i is an integer; The upstream vector n comprises an expression cassette Vn, and the expression cassette Vn comprises sequentially connected: a recombination site (n-1) B, a splice acceptor site n-1, an nth split fragment, a splice donor site n, and a recombination site nA; The downstream vector comprises an expression cassette D*, wherein the expression cassette D* comprises a recombinase gene expression cassette, a recombination site nB, a splice acceptor site n, an n+1 split fragment and a PolyA terminator connected in sequence; The recombinase gene expression cassette includes a promoter, a recombinase gene and a PolyA terminator; The recombination site 1, recombination site 2, ..., and recombination site n are selected from different sites in the same recombination system, and nA and nB can undergo a recombination reaction.

2. The gene delivery vector according to claim 1, wherein (1) When n is equal to 1, in the expression cassette U of the upstream vector, either end of the recombination site 1A is further connected to a recombination site 2A; In the expression cassette D of the downstream vector, either end of the recombinase gene is further connected to a recombination site 2B; (2) When n is equal to 2, in the expression cassette V2# of the upstream vector 2#, either end of the recombination site 2A is further connected to a recombination site 3A; In the expression cassette D# of the downstream vector, either end of the recombinase gene is further connected to a recombination site 3B; (3) When n is an integer ≥ 3, in the expression cassette Vn of the upstream vector n, either end of the recombination site nA is further connected to a recombination site (n+1)A; In the expression cassette D* of the downstream vector, any one end of the recombinase gene is further connected to a recombination site (n+1)B.

3. A gene delivery vector, characterized in that include: n upstream vectors containing target gene split fragments and 1 downstream vector containing target gene split fragments; The target gene includes a first split fragment, a second split fragment, ..., an n-1 split fragment, an nth split fragment and an n+1th split fragment connected sequentially from the 5' end to the 3' end; (1) When n is equal to 1, the upstream vector comprises an expression cassette U', which includes a promoter, a first split fragment, a splice donor site 1, and a recombination site 1A connected in sequence; The downstream vector comprises an expression cassette D', wherein the expression cassette D' comprises a recombination site 1B, a splicing acceptor site 1, a second split fragment, and a PolyA terminator connected in sequence; Either end of the upstream vector expression cassette U' also includes an N-terminal or C-terminal recombinase gene expression cassette, Either end of the downstream vector expression cassette D' further includes a C-terminal or N-terminal recombinase gene expression cassette; The N-terminal recombinase gene expression cassette and the C-terminal recombinase gene expression cassette are respectively located in any two vectors among the n upstream vectors and the 1 downstream vector; The N-terminal recombinase gene expression cassette includes a promoter, an N-terminal recombinase gene fragment-protein splicing donor site and a PolyA terminator; the C-terminal recombinase gene expression cassette includes a promoter, a protein splicing acceptor site-C-terminal recombinase gene fragment and a PolyA terminator; (2) When n is equal to 2, the upstream vector includes upstream vector 1# and upstream vector 2#; the upstream vector 1# contains expression cassette V1#', and the expression cassette V1#' includes a promoter, a first split fragment, a splice donor site 1, and a recombination site 1A connected in sequence; The upstream vector 2#' comprises an expression cassette V2#', wherein the expression cassette V2#' comprises a recombination site 1B, a splice acceptor site 1, a second split fragment, a splice donor site 2, and a recombination site 2A connected in sequence; The downstream vector comprises an expression cassette D#', wherein the expression cassette D#' comprises a recombination site 2B, a splicing acceptor site 2, a third split fragment and a PolyA terminator connected in sequence; Either end of the expression cassette of the upstream vector 1#' or 2#' further includes an N-terminal or C-terminal recombinase gene expression cassette; Either end of the downstream vector expression cassette D' further includes a C-terminal or N-terminal recombinase gene expression cassette; The N-terminal recombinase gene expression cassette and the C-terminal recombinase gene expression cassette are respectively located in any two vectors among the n upstream vectors and the 1 downstream vector; The N-terminal recombinase gene expression cassette includes a promoter, an N-terminal recombinase gene fragment-protein splicing donor site and a PolyA terminator; the C-terminal recombinase gene expression cassette includes a promoter, a protein splicing acceptor site-C-terminal recombinase gene fragment and a PolyA terminator; (3) When n is an integer ≥ 3, the upstream vectors include: upstream vector 1, upstream vector 2, ..., upstream vector n-1 and upstream vector n; The upstream vector 1 comprises an expression cassette V1', and the expression cassette V1' comprises a promoter, a first split fragment, a splice donor site 1, and a recombination site 1A connected in sequence; The upstream vector n-i' comprises an expression cassette Vn-i', wherein the expression cassette Vn-i' comprises sequentially connected: a recombination site (ni-1) B, a splice acceptor site ni-1, the ni-th split fragment, a splice donor site ni, and a recombination site (ni) A; 1≤i≤n-2, i is an integer; The upstream vector n' comprises an expression cassette Vn', and the expression cassette Vn' comprises sequentially connected: a recombination site (n-1) B, a splice acceptor site n-1, an nth split fragment, a splice donor site n, and a recombination site nA; The downstream vector includes an expression cassette D*', and the expression cassette D*' includes a sequentially connected recombination site nB, a splicing acceptor site n, an n+1 split fragment and a PolyA terminator; Any end of the expression cassette of upstream vectors 1 to n further includes an N-terminal or C-terminal recombinase gene expression cassette; Either end of the downstream vector expression cassette D' further includes a C-terminal or N-terminal recombinase gene expression cassette; The N-terminal recombinase gene expression cassette and the C-terminal recombinase gene expression cassette are respectively located in any two vectors among the n upstream vectors and the 1 downstream vector; The N-terminal recombinase gene expression cassette includes a promoter, an N-terminal recombinase gene fragment-protein splicing donor site and a PolyA terminator; the C-terminal recombinase gene expression cassette includes a promoter, a protein splicing acceptor site-C-terminal recombinase gene fragment and a PolyA terminator; The recombination site 1, recombination site 2, ..., and recombination site n are selected from different sites in the same recombination system, and nA and nB can undergo a recombination reaction.

4. The gene delivery vector according to claim 3, characterized in that (1) When n is equal to 1, in the expression cassette U' of the upstream vector, either end of the recombination site 1A is also connected to a recombination site 2A; In the expression cassette D' of the downstream vector, either end of the N-terminal recombinase gene fragment-protein splicing donor site or the protein splicing acceptor site-C-terminal recombinase gene fragment is further connected to a recombination site 2B; (2) When n is equal to 2, in the expression cassette V2#' of the upstream vector 2#, either end of the recombination site 2A is further connected to a recombination site 3A; In the expression cassette D#' of the downstream vector, any one end of the N-terminal recombinase gene fragment-protein splicing donor site or protein splicing acceptor site-C-terminal recombinase gene fragment is further connected to a recombination site 3B; (3) When n is an integer ≥ 3, in the expression cassette Vn' of the upstream vector n, either end of the recombination site nA is further connected to a recombination site (n+1)A; In the expression cassette D*' of the downstream vector, any one end of the N-terminal recombinase gene fragment-protein splicing donor site or protein splicing acceptor site-C-terminal recombinase gene fragment is further connected to a recombination site (n+1)B.

5. The gene delivery vector according to any one of claims 1 to 4, characterized in that The recombination site n and the recombination site n+1 are selected from any one of the recombination systems of Cre / lox, Dre / rox, FLP / FRT, FLPo / FRT, Vika / vox, λ-Int / attP, B2 / B2RT, B3 / B3RT, R / R1RT, T / TDRT, Kw / KwRT, KD / KDRT, ΦC31 / att, γδ / att, Tn3 / att, Gin / att, Bxb1 / att or R4 / att; and n is selected from an integer ≥1.

6. The gene delivery vector according to claim 5, characterized in that The lox sites include but are not limited to loxP, lox71, lox75, lox44, lox66, lox76, lox43, lox72, lox78, lox65, lox511, lox512, lox514, lox5171, lox2272, loxM2, loxM3, loxM7, and loxM11, m2, m3, m7, or m11 variants; The rox sites include but are not limited to roxP, rox1, rox2, rox7, rox8, rox9, rox12, rox34, rox61 or rox85 variants; The FRT sites include but are not limited to FRT, F1, F2, F3, F4 or F5 variants; The att sites include, but are not limited to, attB, attP, attL or attR variants.

7. The gene delivery vector according to claims 1 to 6, characterized in that The sequence length after recombinant upstream and downstream vectors is ≥4.7kb.

8. The gene delivery vector according to any one of claims 1 to 7, wherein The backbone of the gene delivery vector is selected from AAV adeno-associated virus vector, adenovirus vector, HSV vector, poxvirus vector, baculovirus vector, conventional gene expression vector, transposon vector, prokaryotic expression vector, yeast expression vector, zebrafish expression vector, fruit fly expression vector, plant expression vector or nematode expression vector.

9. The gene delivery vector according to any one of claims 1 to 8, characterized in that The promoter includes a ubiquitous expression promoter, a tissue-specific expression promoter or an inducible expression promoter.

10. The gene delivery vector according to any one of claims 1 to 9, characterized in that The terminator includes SV40 pA terminator, rBG pA terminator, BGH pA terminator or hGH terminator.

11. The gene delivery vector according to any one of claims 1 to 10, characterized in that In the expression cassette D, expression cassette D# and / or expression cassette D*, the recombinase gene expression cassette is located at the 3' end of the expression cassette.

12. The gene delivery vector according to any one of claims 1 to 10, characterized in that If the expression cassette D, expression cassette D# and expression cassette D* do not contain a recombinase gene expression cassette, the recombinase gene expression cassette is located at the 3' end and / or 5' end of any one of the expression cassettes: expression cassette U, expression cassette V1#, expression cassette V2#, expression cassette V1, expression cassette Vn-i or expression cassette Vn.

13. The gene delivery vector according to any one of claims 1 to 11, characterized in that Any one of the expression cassettes U, V1#, V2#, V1, Vn-i, and Vn further comprises a recombinase gene expression cassette; the recombinase gene expression cassette is located at the 3' end and / or the 5' end of the expression cassette.

14. A virus, characterized in that The gene delivery vector according to any one of claims 1 to 13 is packaged by packaging cells.

15. The virus according to claim 14, characterized in that The packaging cells include HEK293, HEK293T, HEK293A, HeLa, BHK21, Sf9 and or CHO.

16. Use of the gene delivery vector according to any one of claims 1 to 13, or the virus according to claim 14 or 15 in the preparation of a gene delivery drug, or in the delivery of an ultra-long gene.

17. A gene delivery drug, characterized in that The gene delivery vector comprises the gene delivery vector according to any one of claims 1 to 13.

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