Mini promoter pAPP and application thereof

A promoter and mini technology, applied in the field of neural engineering, can solve the problems of rAAV low targeting, limited functional protein packaging capacity, limited packaging, etc., to achieve the effects of large packaging capacity, improved expression specificity, and high expression efficiency

Pending Publication Date: 2021-11-02
SHENZHEN ENJI BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

First, the maximum packaging capacity of rAAV is 5.2kb, which greatly limits the packaging of large therapeutic gene elements in a single AAV vector; second, the low targeting of rAAV is also a problem that limits the development of rAAV viral tools
The currently commonly used neuron-specific promoter hSyn has a size of 485bp, which further limits the packaging capacity of functional proteins under the premise of a single AAV vector

Method used

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  • Mini promoter pAPP and application thereof
  • Mini promoter pAPP and application thereof
  • Mini promoter pAPP and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1 pAPP gene fragment synthesis

[0034] The pAPP promoter is a partial sequence of the amyloid beta precursor protein (APP, amyloid beta precursor protein) gene. The sequence selects the sequence of the core promoter region from -80 to +40 of the transcription start site of the APP gene, with a total of 120bp The sequence of the promoter is used as the final promoter sequence, and the nucleotide sequence of the promoter pAPP is shown in SEQ ID NO:1.

[0035] The two ends of the promoter pAPP gene fragment were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. after adding restriction endonuclease MluI-HF and BamHI-HF recognition sites.

Embodiment 2

[0036] Example 2 Construction of recombinant adeno-associated virus vector pAAV-pAPP-eYFP

[0037] In this example, because YFP is used as the reporter gene of the promoter, when constructing the recombinant vector, the recombinant adeno-associated vector pAAV-hSyn-eYFP containing the YFP gene is selected as the carrier backbone to connect the pAPP promoter, and the steps are as follows:

[0038] Such as figure 1 As shown, the pAAV-hSyn-eYFP vector was treated with restriction endonucleases MluI-HF and BamHI-HF, and the pAPP fragment was treated with restriction endonucleases MluI-HF and BamHI-HF at the same time, digested at 37°C for 3 hours, The enzyme digestion system is shown in Table 1 and Table 2. After recovering the enzyme digestion product, the ligation premix (2x ligation premix, TAKARA) was used for ligation at 16°C for 30 minutes. The system is shown in Table 3, and the successfully ligated pAAV- pAPP-eYFP vector.

[0039] Table 1 pAAV-hSyn-eYFP vector restrictio...

Embodiment 3

[0046] Example 3 Preparation of recombinant adeno-associated virus AAV-PHP.B-pAPP-eYFP

[0047]In this example, a three-plasmid co-transfection method was used to prepare viruses. Before virus preparation, the plasmids required for packaging viruses need to be extracted using QIAGEN Plasmid Plus Midi Kit (QIAGEN Company, Cat. No. 12943), including recombinant adeno-associated virus vector pAAV-pAPP-eYFP, packaging plasmid AAV-PHP.B and helper plasmids pHelper. Specific steps are as follows:

[0048] Preparation of cells: spread 293T cells in a petri dish containing full medium (DMEM, 10% fetal calf serum, 1% double antibody), and culture in an incubator.

[0049] Prepare transfection reagent: pipette 5.25ml ultrapure water, 75μg packaging plasmid, 75μg recombinant plasmid, 75μg helper plasmid and 800μL 2M calcium chloride solution, and mix gently. Add an equal volume of 2×HBS to the aforementioned reagents, vortex and let stand for 30 minutes.

[0050] Transfected cells: A...

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Abstract

The invention belongs to the technical field of neural engineering, and particularly relates to a mini promoter pAPP for specific efficient expression in neuronal cells. The invention provides a mini promoter pAPP and the application thereof. The nucleotide sequence of the promoter pAPP is as shown in SEQ ID NO: 1. On the other hand, the invention provides the application of the promoter pAPP in a recombinant adeno-associated virus vector, a recombinant adeno-associated virus, a recombinant adeno-associated virus kit, a gene therapy vector and preparation of reagents for controlling nerve cells, gene editing and / or gene therapy. The mini promoter pAPP provided by the invention enables an exogenous gene to be efficiently expressed in neurons in a whole brain range, and has a wide application prospect in the fields of gene editing and gene therapy.

Description

technical field [0001] The invention belongs to the technical field of neural engineering, and in particular relates to a mini-promoter pAPP and its application. Background technique [0002] Adeno-associated virus (AAV) is a non-enveloped, single-stranded DNA virus. Recombinant adeno-associated virus (rAAV) is a recombinant virus modified on the basis of wild-type AAV. Recombinant adeno-associated virus is a widely used gene delivery vector in the field of neuroscience and clinical gene therapy of nervous system diseases due to its advantages of long-term stable expression of genes in mammals, low immunogenicity, and wide host range. [0003] In the process of applying rAAV in neuroscience research and clinical treatment, improving the spatiotemporal specificity and expression level of target gene expression is the key to gene delivery. Strategies to regulate the expression of exogenous genes directly affect the efficiency and safety of gene delivery. For gene therapy of...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/864C12N15/66C12N7/01C12R1/93
CPCC07K14/47C12N15/86C12N15/66C12N7/00C12N2750/14121C12N2750/14143Y02A50/30
Inventor 路中华陈晔菲李田
Owner SHENZHEN ENJI BIOTECHNOLOGY CO LTD
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