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Adeno-associated virus vector targeting cardiac vascular endothelium and application thereof

A cardiovascular and viral vector technology, applied in the direction of virus/bacteriophage, application, virus, etc., can solve the problem of optimizing cardiovascular endothelial transduction, which has not been used yet

Active Publication Date: 2020-10-30
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These two library construction methods combined with in vivo screening have yielded many improved AAV vectors, however, they have not been used to optimize AAV transduction of cardiovascular endothelium in vivo

Method used

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  • Adeno-associated virus vector targeting cardiac vascular endothelium and application thereof
  • Adeno-associated virus vector targeting cardiac vascular endothelium and application thereof
  • Adeno-associated virus vector targeting cardiac vascular endothelium and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 In vivo directed evolution process and screening results

[0041] (1) In order to insert a random heptapeptide into the R588 site of the AAV2 capsid protein gene to construct a polypeptide display library, it is first necessary to use PCR to introduce a stuffer sequence at the R588 site and mutate the relevant sites. The primers are as follows:

[0042] 588-For:5`- GGCCCAGGCGGCC -ACCGCAGATGTCAACACACAAGGC-3`; (SEQ ID NO: 7)

[0043] 588-Rev:5`- TTGGCCTCTCTG - GCCTCTCTGGAGGTTGGTAGATAC-3`; (SEQ ID NO: 8)

[0044] Amp-1: 5`-CGTTGTCAGAAGTAAGTTGGCCGCA-3`; (SEQ ID NO: 9)

[0045] Amp-2: 5`-ATCGGAGGACCGAAGGAGCTAACCG-3`; (SEQ ID NO: 10)

[0046] Underscores are inserted padding sequences. Using the XX2 plasmid (from the University of North Carolina, USA) as a template, the 588-For and Amp-1 primers amplified a 3.1kb fragment, and the 588-Rev and Amp-2 primers amplified a 5.3kb fragment. The fragment amplified by 588-For / Amp-1 was treated with T4 polynucleotide ...

Embodiment 2

[0062] Example 2 Verification of the ability of EC71 and EC73 to transduce cardiac endothelial cells

[0063] The calcium phosphate method was used to transfect 293 cells to produce EC71, EC73, AAV1, and AAV2-Flt1-luc recombinant viruses, in which Flt1 is a promoter specifically expressed in endothelial cells, and luc is a luciferase reporter gene. The virus production process is as follows:

[0064] Passage 293 cells to a 15cm cell culture dish at a ratio of 1:3. After 24 hours, the cell density is about 80%. Add the adenovirus Phelper plasmid, packaging plasmid and recombinant AAV plasmid to 0.25M CaCl at a ratio of 4:1:3. 2 In total, 50μg plasmid and 2ml CaCl are needed per dish of cells 2 , and then the plasmid-CaCl 2 The mixture was added dropwise into an equal volume of 2×HBS for transfection of 293 cells. Change the medium after 8 hours, harvest 30 dishes of cells after 56 hours, centrifuge at 2500rpm for 10min, resuspend the pellet in 10ml DMEM for cesium chloride d...

Embodiment 3

[0068] Example 3 Transduction of EC71 and EC73 to vascular endothelium in mice

[0069] With EC71 in embodiment 2, EC73, AAV1-Flt1-luc recombinant virus is injected 3 * 10 with tail vein 11 The vector copy number reached 7-week-old male C57BL / 6 mice, and a group of mice were sacrificed at 1 week, 2 weeks, 1 month, 2 months, and 4 months, and the hearts, livers, and Luciferase activity was detected in four tissues, brain and lung. The result is as Figure 6 As shown, in the heart, the three viruses can maintain transgene expression for at least 4 months, and the expression levels gradually increase, and always maintain EC71>EC73>AAV1; The expression levels of EC71 and EC73 in the liver remained at a very low level; in the brain, the three viruses were stably expressed within 4 months; It gradually increased before, and then showed a downward trend. Therefore, EC71 and EC73 are able to maintain more efficient and stable transgene expression in the cardiac vascular endotheliu...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to an adeno-associated virus vector for targeting cardiac vascular endothelium and an application of the adeno-associated virus vector. The invention aims to solve the problem that natural AAV has poor cardiovascular endothelial transduction capacity in vivo. The invention provides an adeno-associated virus vector targeting heart vascular endothelium. The library is constructed by inserting an R588 site of an AAV2 capsid protein gene into a coding sequence of random heptapeptide, and inserting the coding sequenceinto a plasmid skeleton containing an AAV2 rep gene and ITR through HindIII / NotI double enzyme digestion. After two rounds of in-vivo directed evolution screening, the two AAV variants EC71 and EC73are obtained, the transduction capacity of AAV to the heart vascular endothelium in vivo is improved, meanwhile, transduction to the liver is greatly reduced, and transgenic expression is maintained for at least four months in the mouse heart vascular endothelium. The EC71 vector is used for conveying the eNOS gene into a myocardial infarction mouse body, so that the activity of the eNOS protein of the heart and the lung is effectively improved, and the EC71 vector has a certain application prospect in gene therapy of cardiovascular endothelial related diseases.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an adeno-associated virus vector targeting cardiac vascular endothelium and application thereof. Background technique [0002] Adeno-associated virus (adeno-associated virus, AAV) has a non-enveloped icosahedral capsid of about 22nm, and its genome is a linear single-stranded DNA of about 4.7kb. Contaminants of the virus Ad5 have been identified for the first time. The replication of AAV needs to be completed with the help of a helper virus, such as adenovirus or herpes simplex virus. The AAV genome mainly encodes two genes, namely rep and cap, both ends of which are composed of terminal repeat sequences ITR. The rep gene uses the two promoters p5 and p19 to encode four non-structural proteins, which are named Rep78, Rep68, Rep52 and Rep40 according to their molecular weight, and are related to viral replication, transcription regulation, genome envelope and integration...

Claims

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Application Information

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IPC IPC(8): C12N15/864C07K7/06C07K14/015
CPCC12N15/86C07K7/06C07K14/005C12N2750/14122C12N2750/14143
Inventor 杨林刘运波魏于全
Owner SICHUAN UNIV
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