Preparation and purification method of exosome-coated AAV vector

A purification method and exosome technology, which is applied in the field of preparation and purification of exosome-wrapped AAV vectors, can solve the problems of inability to enhance retinal tissue transfection and low transfection efficiency of target genes, and achieve the goal of enhancing transfection and improving transfection efficiency Effect

Inactive Publication Date: 2021-04-16
THE SECOND PEOPLES HOSPITAL OF SHENZHEN
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Problems solved by technology

[0006] In the prior art, there is no method for preparing and purifying the AAV target gene carrier encapsulated by exosomes. In the prior art, the AAV target gene carrier is repeatedly pressed, which cannot enhance the transfer in retinal tissue, and the target gene transfection efficiency is low

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  • Preparation and purification method of exosome-coated AAV vector

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Embodiment Construction

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below. The embodiments of the present invention and all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

[0024] The present invention provides a technical solution: a method for preparing an exosome-wrapped AAV vector, comprising the following steps:

[0025] Step 1: Culture Expi293F cells with DMEM complete medium, and use for transfection when the cells grow to a confluence of 3–5×106 living cells / mL, and replace the old culture medium with DMEM basal medium 1-4 hours before transfection base;

[0026] Step 2: Co-transfect the pAAV-target gene plasmid, pAAV-RC2 plasmid and pHelper plasmid into the Expi293F cell culture medium obtained in Step 1. After co-transfection at 37°C for 12-16 hours, add exosome-free fetal bovine serum and continue Cultivate fo...

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Abstract

The invention belongs to the technical field of gene drug vectors, and discloses an exosome-coated AAV vector, an AAV target gene vector, and preparation methods and applications thereof. The method comprises the following steps: culturing Expi293F cells in a DMEM complete medium,carrying out transfection when the cells grow until the fusion degree reaches 3-5 * 10 < 6 > viable cells / mL, and replacing an old culture medium with a DMEM basal culture medium 14 hours before transfection; and co-transfecting pAAV-target gene plasmids, pAAV-RC2 plasmids and pHelper plasmids into the Expi293F cell culture solution, after co-transfecting at 37 DEG C for 12-16 hours, adding exosome-free fetal calf serum, continuously culturing for 48-72 hours, collecting supernatant,carrying out centrifugal separation to obtain the exosome-coated AAV vector. The exosome-coated AAV target gene vector is applied to preparation of drugs for treating diseases corresponding to target genes. The exosome prepared by the invention wraps the AAV target gene vector and is purified, and compared with the AAV target gene vector, the natural membrane structure of the exosome can effectively enhance the transfer of AAV in retinal tissues and improve the target gene transfection efficiency.

Description

technical field [0001] The invention relates to the field of gene drug carriers, in particular to a method for preparing and purifying exosome-wrapped AAV vectors. Background technique [0002] Adeno-associated virus (adeno-associated virus, AAV) is a kind of single-stranded linear DNA defective virus. Its genomic DNA is less than 5kb, without envelope, and its appearance is naked icosahedral particles. AAV cannot replicate independently, and can only replicate and cytolytic infection in the presence of helper viruses (such as adenovirus, herpes simplex virus, vaccinia virus), otherwise only lysogenic latent infection can be established. [0003] Adeno-associated virus vector: The most commonly used adeno-associated virus vector is AAVHelper-FreeSystem, which is a packaging system that does not require an adenovirus helper vector. [0004] Exosomes refer to small membrane vesicles (30-150nm) that contain complex RNA and proteins. Today, they specifically refer to discoid v...

Claims

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Application Information

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IPC IPC(8): C12N15/864C12N15/87
Inventor 段莉梁宇杰徐晓徐丽梅
Owner THE SECOND PEOPLES HOSPITAL OF SHENZHEN
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