Method for efficiently infecting T cells in vitro by virtue of AAV6-type adeno-associated virus

Abstract

The invention discloses a method for efficiently infecting T cells in vitro by virtue of an AAV6-type adeno-associated virus. The method specifically comprises the steps of packaging the AAV6-type adeno-associated virus by virtue of an AAV plasmid, a pHelper plasmid and a pRC plasmid under the action of an infection aid VGF-001, harvesting the virus, carrying out purification and concentration, injecting the T cells by virtue of the prepared AAV6-type adeno-associated virus, and detecting the infection efficiency of the cells by virtue of combination of an immunofluorescence method and a flow cytometry. A result shows that by infecting the human T cells in vitro by virtue of the AAV6 virus, the efficient infection of the human T cells is realized, the infection efficiency can reach 80%-90% or above and is incomparable with other gene transfection methods, a big problem is solved for scholars of in-vitro research of the T cells, and a firm foundation is laid for the clinical research of cell therapy.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and cell engineering, and specifically relates to a method for efficiently infecting T cells with AAV6 type adeno-associated virus in vitro. Background technique [0002] T cells, a kind of lymphocytes, are derived from bone marrow and mature in the thymus. Mature T cells are distributed to the thymus-dependent areas of peripheral immune organs through the bloodstream and settle in them, playing an important role in cellular immunity and immune regulation. With the deepening of cellular immunotherapy, the genetic engineering of T cells is particularly important. [0003] The genetic engineering of T cells refers to the efficient introduction of therapeutically significant genes into T cells, so that T cells can play a role after relevant expression. Taking the application of modified T cells for tumor treatment as an example, the tumor target protein monoclonal antibody is expressed o...

AI Technical Summary

Problems solved by technology

However, the transfection operation of T cells is extremely difficult, and the DNA expression plasmid can hardly be transfected. Even the virus-mediated gene transfection operation has multiple difficulties, as follows: 1) Use adenovirus for transfection: Ad5 type Adenovirus infects cells by binding the Fiber region on the surface of the virus particle to the CAR receptor on the cell, but the surface of T cells lacks CAR receptors, so Ad5 adenovirus has almost no ability to infect T cells

Although some researchers have achieved chimeric transformation of the Ad5-type adenovirus Fiber region, the infection effect on T cells has not been substantially improved.

In addition, the titer of adenovirus is not too high, and the immunogenicity is high. T cells cultured in vitro are very sensitive and fragile, and adenovirus infection will have a great impact on the state of T cells; in addition, T cells are suspension cells, further increasing The difficulty of effectively infecting the virus

2) Use lentivirus and retrovirus for transfection: ① Inactive T cells have no proliferative ability, ② T cell surface receptors are unstable, ③ T cells have a mechanism that hinders reverse transcription, and ④ Retroviruses require T cells to be in Dividing state, but it will cause T cells to enter the late differentiation stage, thus affecting the effect of subsequent cell therapy

However, there are relatively few studies on the high-efficiency infection of T cells by AAV in vitro. Therefore, providing a method for high-efficiency infection of T cells by AAV in vitro has become an urgent problem to be solved at this stage.

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