Muscle-specific promoter for enhancing gene expression

Abstract

The present disclosure relates to a novel polynucleotide of a muscle-specific promoter. In addition, the disclosure also relates to nucleic acid constructs, vectors, viral particles and compositions comprising the polynucleotides described in the disclosure for driving transcription expression of muscle-specific genes, and to the preparation of gene therapies for the treatment of related diseases.

Description

Technical Field

[0001] The present disclosure belongs to the field of biomedicine, and in particular relates to a muscle-specific promoter for enhancing gene expression. Background Art

[0002] Gene therapy is a method of treating certain diseases by transferring therapeutic genetic material to target cells. In recent years, gene therapy research has developed rapidly and has performed well in many rare diseases, making it an ideal treatment method. The three elements of gene therapy include target cells, nucleic acids, and transfer methods. Therefore, in the process of gene therapy, choosing a suitable vector and controllable and effective expression is one of the keys to success. Controllable and effective expression requires a promoter with high specificity and strength. This patent provides a muscle-specific promoter that can be used for gene therapy, which can selectively target muscle tissue, providing options for the application of gene therapy in muscle-related diseases. Summary of the invention

[0003] The present disclosure provides a polynucleotide for enhancing muscle-specific gene expression, wherein the polynucleotide comprises a sequence selected from SEQ ID NO: 1. In one embodiment, the sequence of the polynucleotide is as shown in SEQ ID NO: 1.

[0004] The present disclosure provides a nucleic acid construct comprising: 1) a polynucleotide of interest; 2) a transcriptional regulatory element operably linked to the polynucleotide sequence of interest, wherein the transcriptional regulatory element comprises the polynucleotide as described above, and the transcriptional regulatory element is located upstream of the polynucleotide sequence of interest, and the polynucleotide of interest is a muscle-specific gene.

[0005] The present disclosure provides a vector, which comprises the polynucleotide or the nucleic acid construct as described above.

[0006] In some embodiments, the vector is a viral vector.

[0007] In some embodiments, the vector is an adeno-associated virus (AAV) vector, an adenoviral vector, or a lentiviral vector.

[0008] The present disclosure provides an AAV particle, which comprises the vector and capsid protein as described above.

[0009] The AAV is selected from any AAV serotype isolated from human and non-human mammals or their variants.

[0010] wherein the AAV is selected from the group consisting of serotypes 1, 2, 3, 3B, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, rh10 and hu37.

[0011] The present disclosure provides a pharmaceutical composition, which includes the polynucleotide as described above, the nucleic acid construct as described above, the vector as described above, or the AAV particle as described above, and a pharmaceutically acceptable excipient.

[0012] On the other hand, the present disclosure provides use of the polynucleotide as described above, the nucleic acid construct as described above, the vector as described above, or the AAV particle as described above, or the pharmaceutical composition as described above in the preparation of a medicament for treating or preventing a disease in a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The present disclosure can be more fully understood with reference to the following drawings.

[0014] Figure 1 A dual fluorescence screening system plasmid containing the SCC48 promoter is shown.

[0015] Figure 2 The pIDL-SCC48 plasmid map is shown.

[0016] Figure 3 The pIDL-CK8e plasmid map is shown.

[0017] Figure 4 A flowchart of the construction of pIDL-SCC48 is shown.

[0018] Figure 5 The results of dual fluorescence detection of different cells transfected with pIDL-CK8e / SCC48 are shown.

[0019] Figure 6 In vivo imaging results of 4A-SCC48 BALB / cJ IV 1E10 vg / mouse at 1 and 4 weeks are shown.

[0020] Figure 7 In vivo imaging results of 4A-CK8e BALB / cJ IV 1E10 vg / mouse at 1 and 4 weeks are shown. DETAILED DESCRIPTION

[0021] The following description of the present disclosure is intended only to illustrate various different embodiments of the present disclosure. Therefore, the specific modifications discussed should not be interpreted as limiting the scope of the present disclosure. It is obvious to those skilled in the art that various different equivalents, changes and modifications can be made without departing from the scope of the present disclosure, and it should be understood that these equivalent embodiments will be included herein. All references cited herein, including publications, patents and patent applications, are incorporated herein by reference in their entirety.

[0022] Polynucleotide

[0023] As used herein, the terms "nucleotide" and "polynucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising, consisting essentially of, or consisting of purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases.

[0024] The term "promoter" as used herein means a control sequence, which is a region of a polynucleotide sequence that controls the initiation and rate of transcription of a coding sequence, such as a gene or a transgene. Promoters can be constitutive, inducible, repressible or tissue-specific.

[0025] The term "vector" as used herein refers to a nucleic acid comprising, consisting essentially of, or consisting of a complete replicon, such that the vector can be replicated when placed in a cell by, for example, a transfection, infection, or transformation process. It should be understood in the art that once inside a cell, the vector can be replicated as an extrachromosomal (episomal) element, or can be integrated into a host cell chromosome. The vector may include nucleic acids derived from retroviruses, adenoviruses, herpes viruses, baculoviruses, modified baculoviruses, papillomaviruses, AAV viral vectors, lentiviral vectors, adenoviral vectors, alphaviral vectors, etc. Preferably, the vector described herein is selected from an AAV vector, an adenoviral vector, or a lentiviral vector.

[0026] The term "adeno-associated virus" or "AAV" as used herein refers to members of the class of viruses associated with that name and belonging to the genus Dependoviridae of the family Parvoviridae. Adeno-associated virus is a single-stranded DNA virus that grows exclusively in cells, where certain functions are provided by a co-infecting helper virus. All AAV serotypes apparently exhibit very similar replication characteristics mediated by homologous rep genes; and all carry three related capsid proteins. At least 13 sequentially numbered naturally occurring AAV serotypes are known in the art. Non-limiting exemplary serotypes for use in the methods disclosed herein include any of these 13 serotypes, such as AAV2, AAV8, AAV9. AAV particles comprise, consist essentially of, or consist of three major viral proteins, VP1, VP2, and VP3. In an embodiment, the AAV comprises an AAV capsid protein selected from the group consisting of AAVPHP.B, AAVrh74, AAV110, AAV204, AAV214, AAV214A, AAV214e, AAV214e8, AAV214e9, AAV214e10, AAV ITB102-45, and AAV214AB. In an embodiment, AAV refers to serotype AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13, AAVrh10, AAVhu37, or any one of the AAV serotypes isolated from human and non-human mammals, or variants thereof. In an embodiment, the AAV particles comprise an AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV17, AAV18, AAV19, AAV20, AAV21, AAV22, AAV23, AAV24, AAV25, AAV26, AAV27, AAV28, AAV29, AAV30, AAV31, AAV32, AAV33, AAV34, AAV35, AAV36, AAV37, AAV38, AAV39, AAV40, AAV41, AAV42, AAV43, AAV44, AAV45, AAV46, AAV47, AAV48, AAV49, AAV50, AAV51, AAV52, AAV53, AAV5 V16.3, AAV24.1, AAV27.3, AAV42.12, AAV42-1b, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV 42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5,AAV44.1、AAV44.2、AAV44.5、AAV223.1、AAV223.2、AAV223.4、AAV223.5、AAV223.6、AAV223.7、AAV1-7 / rh.48、AAV1-8 / rh.49、AAV2-15 / rh.62、AAV2-3 / rh.61、AAV2-4 / rh.50、AAV2-5 / rh.51、AAV3.1 / hu.6、AAV3.1 / hu.9、AAV3-9 / rh.52、AAV3-11 / rh.53、AAV4-8 / r11.64、AAV4-9 / rh.54、AAV4-19 / rh.55 ,AAV5-3 / rh.57,AAV5-22 / rh.58,AAV7.3 / hu.7,AAV16.8 / hu.10,AAV16.12 / hu.11,AAV29.3 / bb.1,AAV29.5 / bb.2,AAV106.1 / hu.37,AAV114.3 / hu.40,AAV127.2 / hu.41,AAV127.5 / hu.42,AAV128.3 / hu.44,AAV130.4 / hu.48,AAV145.1 / hu.53,AAV145.5 / hu.54,AAV145.6 / hu.55,AAV161.10 / hu.60,AA V161.6 / hu.61、AAV33.12 / hu.17、AAV33.4 / hu.15、AAV33.8 / hu.16、AAV52 / hu.19、AAV52.1 / hu.20、AAV58.2 / hu.25、AAV3.3、AAV3.4、AAVA3.5 3.7、AAVC1、AAVC2、AAVC5、AAV-DJ、AAV-DJ8、AAVF3、AAVF5、AAVH2、AAVrh.72、AAVhu.8、AAVrh.68、AAVrh.70、AAVpi.1、AAVpi.3、AAVpi.2、AAVrh.60、AAVrh. AVrh.44、AAVrh.65、AAVrh.55、AAVrh.47、AAVrh.69、AAVrh.45、AAVrh.59、AAVhu.12、AAVH6、AAVLK03、AAVH-1 / hu.1、AAVH-5 / hu.3、AAVLG-10 / rh.40 AAVLG-4 / rh.38、AAVLG-9 / hu.39、AAVN721-8 / rh.43、AAVCh.5、AAVCh.5R1、AAVcy.2、AAVcy.3、AAVcy.4、AAVcy.5、AAVCy.5R1、AAVCy.5R2、AAVCy.5R3AAVCy.5R4、AAVcy.6、AAVhu.1、AAVhu.2、AAVhu.3、AAVhu.4、AAVhu.5、AAVhu.6、AAVhu.7、AAVhu.9、AAVhu.10、AAVhu.11、AAVhu.13、AAVhu.15、AAVhu.16、AAVhu.17、AAVhu.18、AAVhu.20、AAVhu.21、AAVhu.22、AAVhu.23.2、AAVhu.24、AAVhu.25、AAVhu.27、AAVhu.28、AAVhu.29、AAVhu.29R、AAVhu.31、AAVhu.32、AAVhu.34、AAVhu.35、AAVhu.37、AAVhu.39、AAVhu.40、AAVhu.41、AAVhu.42、AAVhu.43、AAVhu.44、AAVhu.44R1、AAVhu.44R2、AAVhu.44R3、AAVhu.45、AAVhu.46、AAVhu.47、AAVhu.48、AAVhu.48R1、AAVhu.48R2、AAVhu.48R3、AAVhu.49、AAVhu.51、AAVhu.52、AAVhu.54、AAVhu.55、AAVhu.56、AAVhu.57、AAVhu.58、AAVhu.60、AAVhu.61、AAVhu.63、AAVhu.64、AAVhu.66、AAVhu.67、AAVhu.14 / 9、AAVhu.t 19、AAVrh.2、AAVrh.2R、AAVrh.8、AAVrh.8R、AAVrh.10、AAVrh.12、AAVrh.13、AAVrh.13R、AAVrh.14、AAVrh.17、AAVrh.18、AAVrh.19、AAVrh.20、AAVrh.21、AAVrh.22、AAVrh.23、AAVrh.24、AAVrh.25、AAVrh.31、AAVrh.32、AAVrh.33、AAVrh.34、AAVrh.35、AAVrh.36、AAVrh.37、AAVrh.37R2、AAVrh.38、AAVrh.39、AAVrh.40、AAVrh.46、AAVrh.48、AAVrh.48.1、AAVrh.48.1.2、AAVrh.48.2、AAVrh.49、AAVrh.51、AAVrh.52、AAVrh.53、AAVrh.54、AAVrh.56、AAVrh.57、AAVrh.58、AAVrh.61、AAVrh.64、AAVrh.64R1, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533A mutant, AAAV, BAAV, goat AAV, bovine AAV, AAVhE1.1, AAVhEr1.5, AAVhER1.14, AAVhEr1.8, AAVhEr1.16, AAVhEr1.18, AAVhEr1.35, AAVhEr1.7, AAVhEr1.36, AAVhEr2.29, AAVhEr2.4, AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhER1.23, AAVhEr3.1, AAV2.5T, AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC12, AAV-2-pre-miRNA-101, AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2, AAV Shuffle 100-1, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAVShuffle 100-2, AAV SM 10-1, AAV SM 10-8, AAV SM 100-3, AAV SM 100-10, BNP61AAV, BNP62 AAV, BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.11, AAVhu.53, AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29AAV128.1 / hu.43、Teletype(true type)AAV(ttAAV)、UPENN AAV 10、Teletype AAV 10、AAV CBr-7.1、AAV CBr-7.10、AAV CBr-7.2、AAV CBr-7.3、AAV CBr-7.4、AAV CBr-7.5、AAV CBr-7.7、AAV CBr-7.8、AAV CBr-B7.3、AAV CBr-B7.4、AAV CBr-E1、AAV CBr-E2、AAV CBr-E3、AAV CBr-E4、AAVCBr-E5、AAV CBr-e5、AAV CBr-E6、AAV CBr-E7、AAV CBr-E8、AAV CHt-1、AAV CHt-2、AAVCHt-3、AAV CHt-6.1、AAV CHt-6.10、AAV CHt-6.5、AAV CHt-6.6、AAV CHt-6.7、AAV CHt-6.8、AAV CHt-P1、AAV CHt-P2、AAV CHt-P5、AAV CHt-P6、AAV CHt-P8、AAV CHt-P9、AAVCKd-1、AAV CKd-10、AAV CKd-2、AAV CKd-3、AAV CKd-4、AAV CKd-6、AAV CKd-7、AAV CKd-8、AAV CKd-B1、AAV CKd-B2、AAV CKd-B3、AAV CKd-B4、AAV CKd-B5、AAV CKd-B6、AAV CKd-B7、AAV CKd-B8、AAV CKd-H1、AAV CKd-H2、AAV CKd-H3、AAV CKd-H4、AAV CKd-H5、AAV CKd-H6、AAV CKd-N3、AAV CKd-N4、AAV CKd-N9、AAV CLg-F1、AAV CLg-F2、AAV CLg-F3、AAV CLg-F4、AAV CLg-F5、AAV CLg-F6、AAV CLg-F7、AAV CLg-F8、AAV CLv-1、AAV CLv1-1、AAV CLv1-10、AAV CLv1-2、AAV CLv-12、AAV CLv1-3、AAV CLv-13、AAV CLv1-4、AAV Clv1-7、AAV Clv1-8、AAV Clv1-9、AAV CLv-2、AAV CLv-3、AAV CLv-4、AAV CLv-6、AAV CLv-8、AAV CLv-D1、AAVCLv-D2、AAV CLv-D3, AAV CLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, AAVCLv-E1, AAV CLv-K1, AAV CLv-K3, AAV CLv-AAV-AAV6 CLv-L4, AAV CLv-L5, AAV CLv-L6, AAVCLv-M1, AAV CLv-M11, AAV CLv-M2, AAV CLv-M5, AAV CLv-M6, AAV CLv-M7, AAV CLv-M8、AAVCLv-M9、AAV CLv-R1、AAV CLv-R2、AAV CLv-R3、AAV CLv-R4、AAV CLv-R5、AAV CLv-R6、AAVCLv-R7、AAV-RAV-VCLv、AAV CLv-R9、AAV CSp-1、AAV CSp-10、AAV CSp-11、AAV CSp-2、AAVCSp-3、AAV CSp-4、AAV CSp-6、AAV CSp-7、AAV CSp-8CSp-8、AAV AAV. CSp-8.2、AAVCSp-8.4、AAV CSp-8.5、AAV CSp-8.6、AAV CSp-8.7、AAV CSp-8.8、AAV CSp-8.9、AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1 / HSC1, AAV F11 / HSC11、AAVF12 / HSC12、AAVF13 / HSC13、AAVF14 / HSC14、AAVF15 / HSC1 5、AAVF16 / HSC16、AAVF17 / HSC17、AAVF2 / HSC2、AAVF3 / HSC3、AAVF4 / HSC4 、AAVF5 / HSC5、AAVF6 / HSC6、AAVF7 / HSC7、AAVF8 / HSC8、AAVF9 / HSC9、AAV- PHP.B(PHP.B)、AAV-PHP.A(PHP.A)、G2B-26、G2B-13、TH1.1-32、TH1.1-35、AAVPHP.B2、AAVPHP.B3、AAVPHP.N / PAHP.B-DGTAAHPHP. .B-GGT、AAVPHP.B-ATP、AAVPHP.B-ATT-T、AAVPHP.B-DGT-T、AAVPHP.B-GGT-T、AAVPHP.B-SGS、AAVPHP.B-AQP、AAVPHP.B-QQP、AAVPHP.B-NPHP(3)AAVPHP.B-SNP, AAVPHP.B-QGT, AAVPHP.B-NQT, AAVPHP.B-EGS, AAVPHP.B-SGN, AAVPHP.B-EGT, AAVPHP.B-DST, AAVPHP.B-DST, AAVPHP.B-STP, AAVPHP.B-PQP, AAVPHP.B-SQP, AAVPHP.B-QLP, AAVPHP.B-TMP, AAVPHP.B-TTP, AAVPHP.S / G2A12, AAVG2A15 / G2A3, AAVG2B4, AAVG2B5 and variants thereof. ,

[0027] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, rats, mice, amphibians, reptiles, etc. Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably. In the present disclosure, the preferred subject is a human.

[0028] As used herein, the term "treatment" refers to administering to a subject an effective amount of a polynucleotide, nucleic acid construct, vector, AAV particle or pharmaceutical composition as described herein, so that the subject has a reduction in at least one symptom of the disease or an improvement in the disease, for example, a beneficial or desired clinical outcome. For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, alleviation of one or more symptoms, reduction in the extent of the disease, stabilization of the disease state (i.e., no deterioration), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial relief or total relief), whether detectable or undetectable. Treatment may refer to prolonging survival compared to the expected survival without treatment. Therefore, those skilled in the art recognize that treatment can improve the disease condition, but may not be a complete cure of the disease. As used herein, the term "treatment" includes prevention. Alternatively, treatment is "effective" in the case of a reduction or cessation of the progression of the disease. "Treatment" may also mean prolonging survival compared to the expected survival without treatment. As used herein, the term "effective amount" means an amount sufficient to achieve the desired effect. In the case of treatment or prevention, the effective amount depends on the type and severity of the disorder in question and the characteristics of the individual subject, such as general health, age, sex, weight and tolerance to the pharmaceutical composition. In gene therapy, for example, in some embodiments, the effective amount is an amount sufficient to modulate some or all of the functions of the relevant gene. In other embodiments, the effective amount of AAV viral particles is an amount sufficient to modulate the expression of the gene in the subject. Those skilled in the art will be able to determine the appropriate amount based on these and other factors.

[0029] The experimental materials and instrument information used in this application are shown in Tables 1 and 2:

[0030] Table 1. Reagents

[0031]

[0032]

[0033] Table 2. Instruments

[0034] Instrument Name Source or Model PCR Amplifier Thermo Gel imaging system BioRad Electric constant temperature water bath Thermofisher Centrifuge Eppendorf Carbon dioxide cell culture incubator Thermofisher ELISA reader spectra Max TD3

[0035] Table 3. Primer information

[0036]

[0037] Example

[0038] In order to enable those skilled in the art to better understand the solutions of the present disclosure, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present disclosure, rather than all of the embodiments.

[0039] Example 1: Construction of a dual fluorescence screening system plasmid containing the SCC48 promoter

[0040] Table 4. Sequence information

[0041]

[0042] pIDL-CK8e is a positive control. The plasmid map of pIDL-CK8e is shown in Figure 3 The construction method is referred to CN115948431A. The construction flow chart of pIDL-SCC48 is shown in Figure 4 , pIDL-SCC48 plasmid map can be found at Figure 2 , the specific steps are as follows:

[0043] 1.1. PCR amplification and synthesis of SCC48

[0044] To amplify and synthesize SCC48, two rounds of amplification are required. The PCR amplification system and procedure are shown in Table 5-7.

[0045] Table 5. SCC48 first round amplification system

[0046] Components Volume (μl) 2×GXL mix 25 SCC48PF1~10(10mM) 1*10 <![CDATA[ddH2O]]> 15

[0047] Table 6. SCC48 second round amplification system (take 1 μl of the first round amplification product as template)

[0048] Components Volume (μl) 2×GXL mix 25 SCC48PF1 (10mM) 1 SCC48PF10 (10mM) 1 Template (first round amplification product) 1 <![CDATA[ddH2O]]> 22

[0049] Table 7. PCR reaction program

[0050]

[0051] After PCR amplification, electrophoresis detection was performed; the 410 bp fragment was recovered from the gel to obtain fragment SCC48.

[0052] 1.2. pIDL-CK8e digestion

[0053] Table 8. Enzyme digestion system

[0054]

[0055]

[0056] The enzyme digestion reaction was carried out at 37°C for 2 h, and the enzyme-digested vector B was recovered from the electrophoresis gel.

[0057] 1.3. Construction of pIDL-SCC48

[0058] 1.3.1pIDL-SCC48 plasmid ligation and transformation

[0059] The restriction endonuclease vector B and the synthetic recovery fragment TTRP13 were homologously recombined. The recombination system is shown in Table 9:

[0060] Table 9. Recombination system

[0061] Components Volume (μl) Enzyme-cut vector B (120 ng / ul) 1 SCC48 (15 ng / ul) 2 5×CEII Buffer 4 EXnase II 2 ddH2O 11

[0062] Recombination was carried out at 37°C for 30 min. According to the transformation process in the instruction manual of 2T1 competent cells, 10 μl of the ligation product was transformed into 2T1 competent cells, coated on kanamycin-resistant plates, and cultured at 37°C overnight.

[0063] 1.3.2 Identification of pIDL-SCC48 plasmid

[0064] Pick the monoclonal plaques cultured overnight and add them to 20 μl sterile water, mix them evenly, take 5 μl of the monoclonal plaque dilution after mixing and shake the bacteria, and take another 5 μl to send directly for sequencing verification; according to the sequencing results, select the correctly sequenced 2# (SCC48), clone and save, and extract the plasmid.

[0065] Example 2: Dual fluorescence system screening

[0066] 2.1. Cell transfection

[0067] C2C12 cells, H9C2(2-1) cells and HepG2 cells were respectively plated in 96-well plates at a cell number of 1E4 / well and cultured in a 37° C. carbon dioxide incubator until the cell density reached 70%-90% for transfection.

[0068] Table 10. Transfection system (per well)

[0069]

[0070] After transfection, the cells were cultured for 48 hours and then fluorescence detection was performed.

[0071] Dual luciferase assay

[0072] Detection was performed using Promega's Dual-Luciferase Reporter 1000 Assay system kit:

[0073] 1. After removing the culture medium in the 96-well plate, wash the cells with PBS and discard the PBS;

[0074] 2. Add 20 μl of 1×PLB lysis buffer to each well and leave at room temperature for 15 minutes to lyse the cells;

[0075] 3. Add 100 μl of LARⅡ to each well and place it in the microplate reader to read the value of Firefly Luciferase;

[0076] 4. After reading, take out the 96-well plate, add 100 μl of Stop&Glo Reagent to each well immediately, and read the value of Renilla Luciferase in the microplate reader;

[0077] 5. Process the data.

[0078] The results of dual fluorescence detection are detailed in Figure 5 , Table 11.

[0079] Table 11. Double fluorescence detection results of different cells transfected with pIDL-CK8e / SCC48

[0080]

[0081]

[0082] Example 3: Screening and verification of mouse animal experiments

[0083] 3.1. The pIDL-SCC48 and pIDL-CK8e plasmids were packaged into 4A-SCC48 and 4A-CK8e using the AAV three-plasmid (pIDL-SCC48 / CK8e, Helper plasmid, MyoAAV-4A plasmid) packaging system.

[0084] 3.1.1 Cell plating

[0085] 24 h before transfection, cells were plated at 6.28E+4 cells / cm2 Pave to 5*Stack The serum concentration of the culture medium is 5%, and the amount of cell culture fluid added is 100 ml / 1*Stack.

[0086] 3.1.2 Cell transfection:

[0087] The cell transfection system was prepared to 10% of the cell culture volume.

[0088] The preparation of transfection plasmid is shown in Table 12-13:

[0089] Table 12. Transfection plasmids

[0090] pIDL-SCC48(GOI) <![CDATA[0.1μg / 150cm 2 ]]> Helper Plasmids <![CDATA[26.56μg / 150cm 2 ]]> MyoAAV-4A Plasmid <![CDATA[16.12μg / 150cm 2 ]]>

[0091] Table 13. Transfection plasmids

[0092] pIDL-CK8e(GOI) <![CDATA[0.1μg / 150cm 2 ]]> Helper Plasmids <![CDATA[26.56μg / 150cm 2 ]]> MyoAAV-4A Plasmid <![CDATA[16.12μg / 150cm 2 ]]>

[0093] The plasmid was added to OPti-MEM medium to prepare 5% of the volume of the cell culture medium, and the mixture was inverted and mixed 10 times. The PEIpro with the same mass as the plasmid was added to OPti-MEM medium to prepare 5% of the volume of the cell culture medium, and the mixture was inverted and mixed 10 times. The PEIpro mixture was added to the plasmid mixture, and the mixture was inverted and mixed 5 times. The mixture was allowed to stand at room temperature for 15 minutes, and 1% FBS was added to DMEM medium. The cell supernatant was discarded, and the transfection system was added. The mixture was cultured in an incubator at 37°C and 5% CO2 for 72 hours.

[0094] 3.1.3 Virus Harvest:

[0095] The virus was harvested, purified, sterilized by 0.45 μM and 0.22 μM filtration, and stored in aliquots.

[0096] 3.2. BALB / cJ mice (6-8 weeks old) were injected with 4A-SCC48 and 4A-CK8e via tail vein (injection volume: 1E10 vg / mouse)

[0097] 3.3. In vivo imaging of mice (week 1 and week 4) and data processing

[0098] The results of mouse in vivo imaging are as follows Figure 6 and Figure 7As shown. 4A-SCC48 packaged with pIDL-SCC48 plasmid can express luciferase normally after injection into mice, and the expression intensity is greater than that of the positive control 4A-CK8e. The expression of 4A SCC48 gradually increases from the early to the late injection period, and may not reach the highest point at the end of the experiment. After the second week, the expression site shifts from the liver to the muscle tissue and continues to be expressed in the muscle tissue, and the expression in the liver disappears. The SCC48 promoter has strong muscle specificity, strong activation intensity, and long-lasting effect, which is consistent with the test results at the cellular level, and can be used in gene therapy programs for muscle-related diseases or systemic diseases.

[0099] Table 14. Plasmid sequence list

[0100]

[0101]

[0102]

[0103] Incorporated by Reference

[0104] Each patent and scientific article mentioned herein is incorporated by reference in its entirety for all purposes.

[0105] Equivalence

[0106] The present disclosure may be embodied in other specific ways without departing from its spirit or essential characteristics. Therefore, the above embodiments should be regarded as illustrative in all cases, rather than limiting the invention described herein. Therefore, the scope of the present disclosure is indicated by the appended claims rather than by the above description, and all changes within the equivalent meaning and scope of the claims are intended to be covered therein.

Claims

1. A polynucleotide, the sequence of which is shown in SEQ ID NO:

1.

2. A nucleic acid construct comprising: 1) a polynucleotide of interest; 2) A transcriptional regulatory element operably linked to a polynucleotide sequence of interest, wherein the transcriptional regulatory element comprises the polynucleotide of claim 1 and is located upstream of the polynucleotide sequence of interest.

3. A vector comprising the polynucleotide according to claim 1 or the nucleic acid construct according to claim 2.

4. The vector of claim 3, wherein the vector is a viral vector.

5. The vector of claim 4, wherein the vector is an adeno-associated virus (AAV) vector, an adenoviral vector or a lentiviral vector. The vector according to claim 5 , comprising the nucleic acid sequence shown in SEQ ID NO:

3.

7. An AAV particle comprising the vector according to any one of claims 3 to 6 and a capsid protein.

8. The AAV particle of claim 7, wherein the AAV is selected from the group consisting of serotypes 1, 2, 3, 3B, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, rh10, and hu37.

9. A pharmaceutical composition comprising the polynucleotide of claim 1, the nucleic acid construct of claim 2, the vector of any one of claims 3 to 6 or the AAV particle of claim 7 or 8, and a pharmaceutically acceptable excipient.

10. Use of the nucleic acid construct of claim 2, the vector of any one of claims 3 to 6, the AAV particle of claim 7 or 8, or the pharmaceutical composition of claim 9 in the preparation of a medicament for treating or preventing a disease in a subject.

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