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

A cardiovascular and viral carrier technology, applied in the direction of virus/bacteriophage, virus, application, etc., can solve problems that have not been used, optimize cardiovascular endothelial transduction, etc., and achieve the effect of enhanced activity

Active Publication Date: 2022-03-08
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 its application
  • Adeno-associated virus vector targeting cardiac vascular endothelium and its application
  • Adeno-associated virus vector targeting cardiac vascular endothelium and its application

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 Image 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; gradually increased before, and then decreased. Therefore, EC71 and EC73 are able to maintain more efficient and stable transgene expression in the cardiac vascular endothelium, which may achie...

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Abstract

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. Aiming at the weak ability of natural AAV to transduce the cardiovascular endothelium in vivo, the present invention provides an adeno-associated virus vector targeting the cardiac vascular endothelium, the library of which is inserted into the R588 site of the AAV2 capsid gene The coding sequence of the peptide was then inserted into the plasmid backbone containing the AAV2 rep gene and ITR through HindIII / NotI double enzyme digestion. After two rounds of in vivo directed evolution screening, the present invention obtained two AAV mutant strains EC71 and EC73, which improved the ability of AAV to transduce the cardiac vascular endothelium in vivo, and at the same time greatly reduced the transduction to the liver, and showed a high degree of transduction in the mouse heart Transgene expression was maintained in the vascular endothelium for at least 4 months. The use of EC71 vector to deliver eNOS gene to myocardial infarction mice effectively improves the activity of heart and lung eNOS protein, and has certain application prospects for 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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Patent Type & Authority Patents(China)
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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