Preparation and purification method of exosome-coated AAV vector

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...

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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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