阅读说明：本技术 用于肝脏中蛋白质表达的重组启动子、载体及其应用 (Recombinant promoter and vector for protein expression in liver and application thereof ) 是由 潘杏 刘光猛 张胜 方文晶 何晓斌 于 2020-12-23 设计创作，主要内容包括：本发明属于基因治疗领域,更具体地,涉及一种用于肝脏中蛋白质表达的重组启动子、载体及其应用。本发明公开的一组调控基因在肝脏系统高表达的重组核酸序列。所述重组调控序列片段与目前所报道的相近尺寸大小的其他序列相比,驱动报告基因和人凝血因子FVIII在肝脏系统中表达能力明显增强,持续高水平表达能力增强,适用于重组腺病毒伴随病毒(rAAV)介导的基因治疗。(The invention belongs to the field of gene therapy, and particularly relates to a recombinant promoter and a vector for protein expression in liver and application thereof. The invention discloses a group of recombinant nucleic acid sequences for regulating and controlling high expression of genes in a liver system. Compared with other sequences with similar sizes reported at present, the recombinant regulatory sequence fragment obviously enhances the expression capability of a driving reporter gene and human coagulation factor FVIII in a liver system, enhances the continuous high-level expression capability, and is suitable for recombinant adenovirus associated virus (rAAV) mediated gene therapy.)

1. A recombinant nucleic acid molecule, comprising:

(a) a first polynucleotide having at least 90% nucleic acid sequence identity to the nucleotide sequence set forth in SEQ ID No.1, or a functional fragment of said first polynucleotide;

the recombinant nucleic acid molecule is capable of promoting transcription of a heterologous polynucleotide in liver tissue of a mammal.

2. The recombinant nucleic acid molecule of claim 1, further comprising:

(b) a second polynucleotide having at least 90% nucleic acid sequence identity to the nucleotide sequence set forth in SEQ ID No.2 or SEQ ID No.3, or a functional fragment of said second polynucleotide;

wherein (b) and (a) are linked 5 '-3'.

3. The recombinant nucleic acid molecule of claim 2, further comprising:

(c) a third polynucleotide having at least 90% nucleic acid sequence identity to the nucleotide sequence set forth in SEQ ID No.4 or SEQ ID No.5, or a functional fragment of the third polynucleotide;

wherein (c), (b) and (a) are connected in the order of 5 '-3'.

4. Use of a recombinant nucleic acid molecule according to any one of claims 1 to 3 as a promoter for protein expression in the liver.

5. The use according to claim 4, as a promoter of protein expression in the liver and driving expression of FVIII coagulation factors in the liver.

6. A vector comprising the recombinant nucleic acid molecule of any one of claims 1 to 3.

7. The vector of claim 6, wherein the vector is a viral vector or a plasmid.

8. The vector of claim 6, wherein the vector is an AAV vector.

9. A mammalian host cell comprising the expression vector of any one of claims 6 to 8.

10. A mammal whose genome comprises the recombinant nucleic acid molecule of any one of claims 1 to 3 or the expression vector of any one of claims 6 to 8.

Technical Field

The invention belongs to the field of gene therapy, and particularly relates to a recombinant promoter and a vector for protein expression in liver and application thereof.

Background

Gene therapy (gene therapy) refers to the introduction of exogenous normal genes into target cells to correct or compensate for diseases caused by gene defects or abnormalities, and finally to achieve the therapeutic goal. Since the 90 s of the 20 th century, gene therapy technology has moved from laboratory research to practical clinical applications. The gene therapy of simple genetic diseases is rapidly expanded to the field of tumor therapy, and the research of the gene therapy of simple genetic diseases as antiviral therapy is deeply researched in recent years. The gene therapy of the liver is to introduce therapeutic genes into the liver, take liver cells as biological reaction cells to carry out therapeutic gene expression, generate therapeutic proteins to enter blood circulation and achieve the purpose of treatment; meanwhile, the liver can be used as a target organ to treat liver diseases. Wilson et al succeeded in clinical trials of liver gene therapy for the first time in 1992, and opened the heading for liver gene therapy by introducing genes for low-density lipoprotein (LDL) receptors into the liver of patients with familial hypercholesterolemia. The liver can be used as a bioreactor of genes and a therapeutic target, and the gene therapy of the liver provides a new therapeutic prospect for diseases such as hepatitis, liver cirrhosis, liver cancer and the like.

Adenovirus-associated viruses (AAV) belong to the parvoviridae, are viruses with small virus particles, replication defects and no envelope, and wild type AAV has not been found to cause diseases to human bodies so far. The artificially modified recombinant AAV (rAAV) has the advantages of good safety, low immunogenicity, wide tissue tropism, no integration to host cell genome and the like, and the use of the rAAV as a gene therapy vector has become a hotspot of gene therapy research in recent years. The recombinant AAV has obvious defects as a gene therapy vector, the loading capacity of the recombinant AAV is small, the upper limit of the single-stranded genome AAV for loading the exogenous gene is 4.7kb, the length of the loaded exogenous gene is further increased, and the integrity of the virus particle package is reduced. Due to the limitation of loading capacity, some larger genes are not suitable for being delivered by rAAV vectors, such as hemophilia A pathogenic gene blood coagulation factor VIII, the cDNA size of which is 7056bp, and the F VIII precursor protein coding 2351aa far exceeds the loading capacity of rAAV.

Aiming at the characteristics that the gene of the blood coagulation factor VIII is large and the AAV vector loaded gene is limited by capacity, Lind P, et al reduce the size of the gene by 40% to 4.4kb (Lind P et al, Eur.J.biochem.232:19-27,1995) by replacing a B structural domain (760-1667aa) of the blood coagulation factor VIII with an adaptor peptide (SQ sequence) of 14 amino acids, wherein the B domain deleted F VIII (BDD-F VIII) is a version which is commonly adopted for expressing the F VIII protein at present; jenny McIntosh, et al enhanced the in vivo expression of this protein by inserting a 17aa short peptide V3(V3-F VIII) containing 6 glycosylation sites in the middle of the SQ sequence (Jenny McIntosh et al, BLOOD.121(17), 2013). However, the size of BDD-FVIII or V3-FVIII is close to the loading upper limit of rAAV, and the space for regulating and controlling the regulatory elements (promoter and Poly A) for regulating and controlling the expression of the target gene is only about 300 bp.

The main synthesis site of F VIII is hepatocyte and hepatic sinus endothelial cell, The current strategy for treating hemophilia A by using rAAV gene is to adopt small-sized liver-specific promoter to assemble promoter, BDD-F VIII or V3-F VIII and polyA on The limited space of The same AAV vector while reducing target gene F VIII, for example, The expression cluster of BMN270 of BIOMARIN company is HLP-BDD F VIII-sPA (clinical trials. gov number, NCT02576795), The expression cluster of SPK-8011 of SPA company is TTRm-BDD F VIII-Rabbet vitreous gene polyA (clinical Tris. gov number, NCT03003533), both of which adopt small-sized liver-specific promoter to drive The expression of target gene, certain effect has been achieved in clinical trials of stage I/I (Rangara jan, The same clinical trial of liver-II, N. and J. 2017. 7. multidot. 20. 7. multidot. 7. multidot. multid, among them, hemophilia a gene therapy drug BMN270 of BIOMARIN has filed a marketing application to EMA. However, due to the restriction of promoter size, there is still a small difference between the promoter strength and some promoters with larger size.

An efficient promoter can continuously and highly start the expression of a target gene, reduce the dosage required for achieving the treatment effect and reduce the administration times, thereby greatly reducing the treatment cost and the immune response of an organism. A liver-specific promoter that is widely known at present is the TBG promoter (Human thyroxine binding globulin promoter, Human thyroxine-binding globulin promoter, Bish et al, 2011; Carrilo-Carrasco et al, 2010; Cotugno et al, 2011; Gao et al, 2002). However, the promoter has not ideal promoter strength in liver and the expression level is not high (CN 111218446A). Thus, various modified novel promoters based on TTR promoter (Transthyretin promoter, thyroxine transporter promoter) and hAAT promoter (Human α 1-antitrypsin promoter) are used in the gene therapy programs for hemophilia A and hemophilia B, including TTRm promoter (TTR promoter after mutation in SPARK corporation SPARK-8011, clinical Trials. gov number, NCT03003533), ApoE-HCR-hAAT promoter (US7351813B2), HLP promoter (promoter in BIOMARIN corporation BMN270, clinical Trials. gov number, NCT 576795), CRM8/TTRp/MVM (mouse TTR promoter region after addition of MVM intron, CRM8 enhancer sequence before promoter region, promoter region in sangomy promoter, mouse promoter 61525, mouse promoter region after addition of MVM intron 201, TTRp promoter region after addition of TTRm 030201, wu Zhijian et al, 2009), ATh1-5 series promoter (CN 111218446A).

Due to the limited loading capacity of rAAV, when loading a larger gene such as blood coagulation factor VIII, the length of the promoter must be reduced, and correspondingly, the key expression regulatory elements contained in the deleted sequence are lost, and the expression strength and specificity of the target gene are reduced. How to maintain the strength and specificity of promoter to start target gene under the condition of maximally reducing the size of promoter is an important problem to be solved when rAAV is used as a vector to deliver larger gene. The liver promoter after the transformation is basically transformed around TTR and hAAT as cores. Therefore, it is difficult to engineer a promoter having very strong expression strength while maintaining a small size. How to jump out of the existing reconstruction is limited, and a small-size promoter with better expression strength is screened, so that the problem that the invention needs to solve is solved when rAAV is used as a vector to deliver larger genes.

Disclosure of Invention

Aiming at the defects or improvement requirements of the prior art, the invention aims to provide a liver-specific promoter with small size and high expression strength, and apply the liver-specific promoter to drive the expression of a larger exogenous gene (such as a FVIII coagulation factor gene), thereby solving the technical problems of limited loading capacity of rAAV vectors used in the prior gene therapy and poor expression strength of the prior liver-specific promoter.

To achieve the above object, the present invention provides a recombinant nucleic acid molecule comprising:

(a) a first polynucleotide having at least 90% nucleic acid sequence identity to the nucleotide sequence set forth in SEQ ID No.1, or a functional fragment of said first polynucleotide;

the recombinant nucleic acid molecule is capable of promoting transcription of a heterologous polynucleotide in liver tissue of a mammal.

Preferably, the recombinant nucleic acid molecule further comprises:

(b) a second polynucleotide having at least 90% nucleic acid sequence identity to the nucleotide sequence set forth in SEQ ID No.2 or SEQ ID No.3, or a functional fragment of said second polynucleotide;

wherein (b) and (a) are linked 5 '-3'.

Preferably, the recombinant nucleic acid molecule further comprises:

(c) a third polynucleotide having at least 90% nucleic acid sequence identity to the nucleotide sequence set forth in SEQ ID No.4 or SEQ ID No.5, or a functional fragment of the third polynucleotide;

wherein (c), (b) and (a) are connected in the order of 5 '-3'.

According to another aspect of the present invention there is provided the use of the recombinant nucleic acid molecule as a promoter for expression of a protein in the liver.

Preferably, the recombinant nucleic acid molecule acts as a promoter for protein expression in the liver to drive expression of FVIII coagulation factors in the liver.

According to another aspect of the present invention, there is provided a vector comprising said recombinant nucleic acid molecule.

Preferably, the vector is a viral vector or a plasmid.

Preferably, the vector is an AAV vector.

According to another aspect of the present invention, there is provided a mammalian host cell comprising said expression vector.

According to another aspect of the invention there is provided a mammal whose genome comprises said recombinant nucleic acid molecule or said expression vector.

Through the technical scheme, compared with the prior art, the invention can obtain the following beneficial effects:

the invention provides a group of small-sized recombinant nucleic acid sequences for regulating high expression of genes in a liver system. Compared with other sequences with similar sizes reported at present, the recombinant regulatory sequence fragment can obviously enhance the expression capacity of a driving reporter gene and human coagulation factor FVIII in a liver system, and is suitable for recombinant adeno-associated virus (rAAV) mediated gene therapy.

Drawings

FIG. 1 is a schematic structural diagram of a PFD-rAAV-CMV-mCHERRY-bGHpA vector;

FIG. 2 is a graph comparing the effect of different promoters on mCHERRY fluorescence in mouse liver tissues;

FIG. 3 is a schematic diagram of the PFD-rAAV-HLP-BDD-FVIIIopt (WJ) -spolyA vector structure;

FIG. 4 is a graph comparing the effect of FVIII expression from different promoters on C57 mice;

FIG. 5 is a graph of the ratio of AVS series promoter to HLP promoter FVIII expression at 100 days post virus injection;

FIG. 6 is a graph of the ratio of AVS series promoter FVIII expression to ATh3 promoter 160 days after virus injection.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The present invention provides a recombinant nucleic acid molecule comprising:

(a) a first polynucleotide having at least 90% nucleic acid sequence identity to the nucleotide sequence set forth in SEQ ID No.1, or a functional fragment of said first polynucleotide; the nucleic acid molecule is capable of promoting transcription of a heterologous polynucleotide in liver tissue of a mammal.

In some embodiments, the recombinant nucleic acid molecule further comprises:

(b) a second polynucleotide having at least 90% nucleic acid sequence identity to the nucleotide sequence set forth in SEQ ID No.2 or SEQ ID No.3, or a functional fragment of said second polynucleotide; wherein (b) and (a) are linked 5 '-3'.

In other embodiments, the recombinant nucleic acid molecule further comprises:

(c) a third polynucleotide having at least 90% nucleic acid sequence identity to the nucleotide sequence set forth in SEQ ID No.4 or SEQ ID No.5, or a functional fragment of the third polynucleotide; wherein (c), (b) and (a) are connected in the order of 5 '-3'.

The invention also provides the application of the recombinant nucleic acid molecule as a liver-specific promoter.

In some embodiments, the promoter can be used, without limitation, to promote expression of the FVIII coagulation factor gene in the liver.

The invention also provides expression vectors comprising the recombinant nucleic acid molecules of the invention, wherein the nucleic acid molecule is operably linked to a heterologous polynucleotide.

In a preferred embodiment, the expression vector is a plasmid or viral vector. Examples of mammalian expression vectors include adenovirus vectors, plasmid vectors of the pSV and pCMV series, vaccinia vectors and retroviral vectors, and baculoviruses. In some embodiments, the expression vector is an adeno-associated viral vector rAAV.

The expression vector may also include one or more of the following elements: an origin of replication, a selectable marker, and a multiple cloning site.

The invention also provides a mammalian host cell comprising an expression vector of the invention. The expression vector may be transfected into the host cell by any suitable method. Preferably, the host cell is a mammalian cell (e.g., a human cell). These host cells may be isolated cells.

The nucleic acid molecule is capable of promoting transcription of an operably linked heterologous polynucleotide in a mammalian cell. Preferred mammalian cells include mouse cells, rat cells, hamster cells, monkey cells, and human cells. Examples of such cells include HEK cells and derivatives (e.g., HEK293T, HEK293A), PerC6 cells, 911 cells, CHO cells, HCT116 cells, HeLa cells, COS cells, and VERO cells; cancer cells such as HepG2, a549, and MCF 7; primary cells isolated from a human or animal biopsy; and stem cells (including pluripotent cells such as embryonic stem cells and Induced Pluripotent Stem (iPS) cells; and pluripotent stem cells such as hematopoietic stem cells, mesenchymal stem cells, and the like).

The invention also provides a mammal whose genome comprises a recombinant nucleic acid molecule of the invention or an expression vector of the invention. Preferably, the nucleic acid molecule of the invention or the expression vector of the invention is inserted into the genome of a mammal such that a heterologous polynucleotide operably linked to said nucleic acid molecule of the invention or operably inserted into said expression vector of the invention is expressed in one or more cells of the mammal. Preferably, the mammal is a mouse or rat. In some embodiments, the mammal is a non-human mammal.

To facilitate a review of various embodiments of the invention, the following provides an explanation of specific terms:

5 'and/or 3': nucleic acid molecules (such as DNA and RNA) have 5 'and 3' ends because mononucleotides react in such a way that the 5 'phosphate of one mononucleotide pentose ring is linked to its adjacent 3' oxygen in one direction by a phosphodiester bond to obtain a polynucleotide. Thus, when its 5 ' phosphate is not linked to the 3 ' oxygen of the pentose ring of a mononucleotide, one end of the linear polynucleotide is referred to as the "5 '" end. When its 3 ' oxygen is not linked to the 5 ' phosphate of the pentose ring of another mononucleotide, the other end of the polynucleotide is called the "3 '" end. Although the 5 'phosphate of one single nucleotide pentose ring is linked to the 3' oxygen of its adjacent ring, the internal nucleic acid sequence may also have 5 'and 3' ends.

In linear or circular nucleic acid molecules, discrete internal elements are referred to as "downstream" or "upstream" or 5 'of 3' elements. For DNA, the term reflects transcription in the 5 'to 3' direction along the DNA strand. Promoters and enhancers, which refer to the transcription of a linked gene, are generally located 5' or upstream of the coding region. However, enhancer elements can exert their effect even when located 3' to the promoter element and coding region. Transcription termination and polyadenylation signals are located 3' or downstream of the coding region.

As used herein, a reference to "at least 90% identity" means "at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity" to a particular reference sequence.

Example 1

Recombinant promoter fragments

Recombinant promoter fragments consisting of the sequences identified in table 1 were generated.

Table 1: sequences of recombinant promoter fragments

Promoter name	The third polynucleotide	Second polynucleotide	A first polynucleotide
				AVS1 promoter	Is free of	Is free of	SEQ ID NO.1
AVS2 promoter	Is free of	SEQ ID NO.2	SEQ ID NO.1
				AVS3 promoter	Is free of	SEQ ID NO.3	SEQ ID NO.1
AVS4 promoter	SEQ ID NO.4	SEQ ID NO.2	SEQ ID NO.1
				AVS5 promoter	SEQ ID NO.5	SEQ ID NO.2	SEQ ID NO.1
AVS6 promoter	SEQ ID NO.4	SEQ ID NO.3	SEQ ID NO.1
				AVS7 promoter	SEQ ID NO.5	SEQ ID NO.3	SEQ ID NO.1

The first, second and third polynucleotides (when present) are linked consecutively in the above promoter fragment. The resulting promoters were designated in order as AVS1, AVS2, AVS2, AVS3, AVS4, AVS5, AVS6, and AVS 7.

Example 2

Animal experiment comparison of mCHERRY fluorescent gene effect initiated by different promoters

1. Construction of rAAV expression vectors comprising different promoters

In order to compare the promoter strengths of different promoters, the chimeric promoter, the reported broad-spectrum strong promoter CMV and the liver-specific promoter TBG are respectively constructed on the AAV vector PFD-rAAV-CMV-mCHERRY-bGHpA, as shown in figure 1.

ITR (inverted terminal repeat) in FIG. 1, inverted terminal repeat of 145bp in length; CMV enhancer/promoter, human cytomegalovirus early promoter; mCHERRY, red luciferase gene reading frame; BGH polyA, polyadenylic acid-micropropagation of bovine growth hormone; amp, reading frame of ampicillin resistance gene; GmR, hygromycin resistance gene reading frame; tn7F/R, Tn7 transposon; ori: a replication initiation site; XhoI, AgeI, SacI, restriction endonuclease sites. The specific construction strategy comprises the following steps.

1.1, carrying out double enzyme digestion on PFD-rAAV-CMV-mCHERRY-bGHpA by using restriction enzymes XhoI and AgeI, and carrying out linearization treatment;

1.2 designing a primer to amplify a required promoter fragment, and reserving a restriction endonuclease site and a homologous recombination arm of about 18bp for recombining with a linearized vector during primer design;

1.3 selecting Clonexpress II One Step Cloning Kit (Novozan, C112) and Clonexpress Multi One Step Cloning Kit (Novozan, C113) according to the number of the recombinant fragments required to carry out homologous recombination on the target fragment and the vector skeleton;

1.4 transforming the homologous recombination product into STBL3 competence, coating LB plate, culturing overnight at 37 ℃, picking single clone for identification, and shaking bacteria to extract plasmid for identification of correct clone.

The promoter level of different promoters is compared according to the promoter mCHERRY fluorescent protein expression. Promoter sequence information is shown in table 2:

TABLE 2 sequence information of different promoters

2. 293T cell packaging rAAV8 virus and purifying

The different promoters, fluorescent proteins mCERRY and bGHpolyA AAV expression vectors constructed in step 1 are packaged into AAV2/8 serotype viruses by a 293 three-plasmid system, and the AAV viruses are purified by iodixanol density gradient ultracentrifugation after precipitation of the viruses by PEG8000 (see Aslanidii et al, 2009, Proc. NatlAcad. Sci. USA,206: 5059-5064). The virus titer was quantified by Q-PCR and silver staining (method reference Lock et al 2010hum Gen Ther.21: 1273-1285).

3. Tail vein injection C57 mouse

Different AAV viruses were injected into C57 mice at the same dose of 3E +13vg/kg by tail vein injection (n ═ 3), 5 weeks after virus injection, cardiac perfusion was performed with physiological saline and 4% paraformaldehyde, and the heart, liver, spleen, lung, kidney, brain and the like were soaked in 4% paraformaldehyde overnight and then in 30% sucrose solution for 72 hours. Taking liver tissue, freezing and slicing at low temperature, and observing fluorescence under a fluorescence microscope. The difference of the different promoters in the promotion of red fluorescence is compared, and the result is shown in FIG. 2.

FIG. 2 is a comparison of the effect of different promoters on mCHERRY fluorescence in mouse liver tissues. AAV viruses of different promoters were injected into C57 mice (n ═ 3) by tail vein injection at the same dose of 3E +13vg/kg, and liver tissue mCHERRY fluorescence intensity was compared 5 weeks after virus injection. The exposure time is about 10ms, about 20, or about 500.

As can be seen from the experimental results of fig. 2, the expression level of the broad-spectrum strong promoter CMV in the liver was very low 5 weeks after AAV virus injection. The commonly used hepatinic promoter, TBG, expresses at a level in the liver that is somewhat stronger than CMV, but there are still no other promoters that have been engineered to be stronger. The expression intensity of AVS1 after AAVS1 modification is almost the same as that of TBG, and the fluorescence intensity of AVS2 and AVS3 is far higher than that of TBG. The result shows that the modified AVS1, AVS2 and AVS3 promoters can highly express fluorescent protein in liver.

Example 3

Comparison of Effect of FVIII factor promoters on different promoters in animal experiments

1. Different promoters for promoting BDD-FVIII vector construction

To further discuss whether the high promoter levels of the AVS1, AVS2, AVS3 promoters in the liver are affected by the downstream linked genes and whether the addition of expression regulatory enhancing elements (AVS4, AVS5, AVS6, AVS7) prior to AVS2, AVS3 can further increase the expression of the gene of interest. Promoter information is shown in table 3.

TABLE 3 sequence information of different promoters

FVIII factor was substituted for reporter gene mCHERRY, and different promoters, BDD-FVIII (B domain deleted FVIII) and sPolya expression cassettes were constructed according to the molecular cloning method in example 2, and constructed on AAV vector PFD-rAAV-HLP-BDD-FVIII-spolyA (FIG. 3), in which PFD-rAAV-HLP-BDD-FVIII-spolyA vector was subjected to double digestion with restriction enzymes XhoI and AscI, followed by linearization treatment.

FIG. 3 is a schematic diagram of the structure of the PFD-rAAV-HLP-BDD-FVIIIopt (WJ) -spolyA vector. Wherein ITR, inverted terminal repeat, length is 145bp inverted terminal repeat; HLP promoter, promoter used by Biomarin for treating hemophilia a project BMN270, BDD-fviiiopt (wj), codon optimized BDD-FVIII; polyA, polyadenylic acid tailing signal; amp, reading frame of ampicillin resistance gene; GmR, hygromycin resistance gene reading frame; tn7F/R, Tn7 transposon; ori: a replication initiation site; XhoI, AscI, restriction enzyme site.

2. Comparison of FVIII expression levels from different promoters

The plasmid constructed in example 1 above was packaged into AAV2/8 serotype virus by sf9 One-bac system. Different AAV viruses were injected into C57 mice (n ═ 6) at the same dose of 5E +12vg/kg tail vein, and FVIII expression levels were measured by orbital venous plexus bleeding ELISA, 70 days, 100 days, 130 days, 160 days after virus injection. The ELISA antibody pair used was F8C-EIA (enzyme research laboratories), a standard curve was drawn using human factor VIII from green crosses, and the concentration of factor VIII was determined using Byunnan BCA protein concentration assay kit (enhanced) (product No. P0010S). The results were plotted and analyzed using Graphpad prism5.0, as shown in fig. 4.

FIG. 4 is a graph comparing the effect of FVIII expression from different promoters in C57 mice. AAV viruses of different promoters were injected into C57 mice (n ═ 6) at the same dose of 5E +12vg/kg by tail vein injection, and the virus was injected for 70 days, 100 days, 130 days, and 160 days, and mouse plasma was collected separately and the FVIII content in the plasma was measured by ELISA.

Unlike patent CN111218446A, the first detection time point is 14 days, the last detection time point is 70 days, the first detection time point is 70 days after virus injection, and the last detection time point is 130 days, so that the promoter strength and the promoter persistence after modification can be better evaluated. Wherein ATh3 is the promoter in patent CN111218446A, the F8 content in the patent at 70 days is 923.03 + -425.98 ng/ml, and as a control, the F8 content in the invention at 70 days is 1118.06 + -125.45 ng/ml. The fluctuation between the two is small, which shows that the detection result in the invention is stable and reliable.

From the overall trend of the detection results, due to the timeliness of AAV expression, the level of FVIII expression of each promoter is reduced to different degrees from day 70 to day 160. Overall, the FVIII-activating ability of AVS1, AVS2, AVS3, AVS4, AVS5, AVS6, AVS7 was significantly better than that of ttam by Spark and HLP promoter by Biomarin. 2way ANOVA analysis in Graphpad prism5.0 software resulted in P and t values between groups as shown in tables 4 to 7:

TABLE 4 comparison of HLP with promoters of the series of the invention

TABLE 5 comparison of TTRm with promoters of the series of the invention

TABLE 6 comparison of CRM8/TTRp/MVM to the series of promoters of the invention

TABLE 7 ATh3 comparison with promoters of the series of the invention

From tables 4-7 above, it can be seen that the 7 newly modified promoters of the invention, AVS1-7, are all significantly superior to HLP and TTRm promoters, with P values <0.05 at almost all time points. When compared with CRM8/TTRp/MVM promoter with the length of 366bp, AVS2, AVS4 and AVS5 all have P values of <0.05 at 2 time points, and AVS6 and AVS7 have P values of <0.05 at 3 time points, and the lengths of the above 5 promoters are all shorter than 366bp, and the longest is AVS 7327 bp. When compared with ATh3 promoter, only AVS6 and AVS7 have P value <0.05 at 2 time points, the promoter strength of AVS6 and AVS7 which are newly modified is also significantly better than that of ATh3 promoter, and the length of AVS6 is 282bp, which is 4bp shorter than 286bp of ATh3 promoter. Further comparison of the fold-relationship between the recombinant promoters provided by the present invention and HLP in promoting FVIII expression is shown in figure 5 and table 8. It can be seen that the promoter level of the AVS series promoter of the present invention is significantly higher than HLP, and all promoters are 4.68 times higher (AVS1) and up to 9.19 times higher (AVS6) than HLP promoter level 100 days after virus injection. FIG. 5 shows the ratio of AVS series promoter expression to HLP promoter FVIII expression at 100 days post virus injection. The average expression level of HLP over 100 days was defined as 1.

TABLE 8 fold relationship between recombinant promoters and HLP provided by the invention in promoting expression of FVIII expression

Days after virus injection	70d	100d	130d	160d
					AVS1/HLP ratio	4.34	4.68	5.77	3.56
AVS2/HLP ratio	4.76	7.47	5.18	4.16
					AVS3/HLP ratio	3.84	5.11	5.30	1.98
AVS4/HLP ratio	0.64	7.26	7.56	3.80
					AVS5/HLP ratio	5.14	6.16	5.47	3.96
AVS6/HLP ratio	7.00	9.19	6.91	5.81
					AVS7/HLP ratio	7.10	7.24	5.77	7.42

Also, fold comparison in FVIII expression level was performed with the small liver promoter ATh3 disclosed in Chinese patent CN111218446A, and the results are shown in FIG. 6 and Table 9. It can be seen that the promoter level of the AVS series promoter of the present invention is still higher than ATh3 as a whole, and 160 days after virus injection, except that AVS3 is 0.68 times (3 other time points are all higher than ATh3) of ATh3, other promoters are all above 1.23 times (AVS1) and up to 2.56 times (AVS 7). Therefore, the length and the expression strength of the liver high-expression promoter provided by the invention are superior to those of other promoters in the field. In the field of gene therapy, AAV vectors or other viral vectors are required to be used for highly expressing exogenous genes in livers, so that stronger small-size high-promoter-effect promoter selection is provided, and the development of the field is remarkably promoted. FIG. 6 is a graph showing the ratio of the expression amount of FVIII from the promoter ATh3 in AVS series at 160 days after virus injection. The average expression level of ATh3 at day 160 was defined as 1.

TABLE 9 fold comparison of promoters of the invention with the liver mini-promoter ATh3 for FVIII expression levels

Days after virus injection	70d	100d	130d	160d
					AVS1/ATh3 ratio	1.63	0.96	1.15	1.23
AVS2/ATh3 ratio	1.78	1.53	1.03	1.44
					AVS3/ATh3 ratio	1.44	1.05	1.06	0.68
AVS4/ATh3 ratio	0.24	7.26	7.56	1.31
					AVS5/ATh3 ratio	1.93	1.26	1.09	1.37
AVS6/ATh3 ratio	2.62	1.88	1.38	2.00
					AVS7/ATh3 ratio	2.66	1.48	1.15	2.56

It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

Sequence listing

<120> recombinant promoter for protein expression in liver, vector and application thereof

<141> 2020-12-22

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 147

<212> DNA

<213> Artificial Sequence

<400> 1

tctgctaatg gactccattt cccagcgctc cccgcgacct gcccagcaca ccctggggca 60

gcgccgtgac gtcagcacgc cgggcgggga ccgggagatc ctataaaatt ggggcggtgg 120

ggggccagcg gcagttcccg gcggccc 147

<210> 2

<211> 63

<212> DNA

<213> Artificial Sequence

<400> 2

aggcaaggtt catatttgtg taggttactt attctccttt tgttgactaa gtcaataatc 60

aga 63

<210> 3

<211> 81

<212> DNA

<213> Artificial Sequence

<400> 3

gggcgactca gatcccagcc agtggactta gcccctgttt gctcctccga taactggggt 60

gaccttggtt aatattcacc a 81

<210> 4

<211> 54

<212> DNA

<213> Artificial Sequence

<400> 4

ccaccccctc caccttggac acaggacgct gtggtttctg agccaggtac aatg 54

<210> 5

<211> 99

<212> DNA

<213> Artificial Sequence

<400> 5

tacctgctga tcgcccggcc cctgttcaaa catgtcctaa tactctgtct ctgcaagggt 60

catcagtagt tttccatctt actcaacatc ctcccagtg 99

<210> 6

<211> 210

<212> DNA

<213> Artificial Sequence

<400> 6

aggcaaggtt catatttgtg taggttactt attctccttt tgttgactaa gtcaataatc 60

agatctgcta atggactcca tttcccagcg ctccccgcga cctgcccagc acaccctggg 120

gcagcgccgt gacgtcagca cgccgggcgg ggaccgggag atcctataaa attggggcgg 180

tggggggcca gcggcagttc ccggcggccc 210

<210> 7

<211> 228

<212> DNA

<213> Artificial Sequence

<400> 7

gggcgactca gatcccagcc agtggactta gcccctgttt gctcctccga taactggggt 60

gaccttggtt aatattcacc atctgctaat ggactccatt tcccagcgct ccccgcgacc 120

tgcccagcac accctggggc agcgccgtga cgtcagcacg ccgggcgggg accgggagat 180

cctataaaat tggggcggtg gggggccagc ggcagttccc ggcggccc 228

<210> 8

<211> 264

<212> DNA

<213> Artificial Sequence

<400> 8

ccaccccctc caccttggac acaggacgct gtggtttctg agccaggtac aatgaggcaa 60

ggttcatatt tgtgtaggtt acttattctc cttttgttga ctaagtcaat aatcagatct 120

gctaatggac tccatttccc agcgctcccc gcgacctgcc cagcacaccc tggggcagcg 180

ccgtgacgtc agcacgccgg gcggggaccg ggagatccta taaaattggg gcggtggggg 240

gccagcggca gttcccggcg gccc 264

<210> 9

<211> 309

<212> DNA

<213> Artificial Sequence

<400> 9

tacctgctga tcgcccggcc cctgttcaaa catgtcctaa tactctgtct ctgcaagggt 60

catcagtagt tttccatctt actcaacatc ctcccagtga ggcaaggttc atatttgtgt 120

aggttactta ttctcctttt gttgactaag tcaataatca gatctgctaa tggactccat 180

ttcccagcgc tccccgcgac ctgcccagca caccctgggg cagcgccgtg acgtcagcac 240

gccgggcggg gaccgggaga tcctataaaa ttggggcggt ggggggccag cggcagttcc 300

cggcggccc 309

<210> 10

<211> 282

<212> DNA

<213> Artificial Sequence

<400> 10

ccaccccctc caccttggac acaggacgct gtggtttctg agccaggtac aatggggcga 60

ctcagatccc agccagtgga cttagcccct gtttgctcct ccgataactg gggtgacctt 120

ggttaatatt caccatctgc taatggactc catttcccag cgctccccgc gacctgccca 180

gcacaccctg gggcagcgcc gtgacgtcag cacgccgggc ggggaccggg agatcctata 240

aaattggggc ggtggggggc cagcggcagt tcccggcggc cc 282

<210> 11

<211> 327

<212> DNA

<213> Artificial Sequence

<400> 11

tacctgctga tcgcccggcc cctgttcaaa catgtcctaa tactctgtct ctgcaagggt 60

catcagtagt tttccatctt actcaacatc ctcccagtgg ggcgactcag atcccagcca 120

gtggacttag cccctgtttg ctcctccgat aactggggtg accttggtta atattcacca 180

tctgctaatg gactccattt cccagcgctc cccgcgacct gcccagcaca ccctggggca 240

gcgccgtgac gtcagcacgc cgggcgggga ccgggagatc ctataaaatt ggggcggtgg 300

ggggccagcg gcagttcccg gcggccc 327