Lentivirus Production For CART Therapy

Cellular therapy known as chimeric antigen receptor (CAR) T cell therapy directs a patient's T cells to specifically target and eradicate tumor cells. CARs are genetically modified fusion proteins made up of two domains: 

(1) An intracellular T cell signaling and costimulatory domain.

(2) An antigen recognition domain derived from a monoclonal antibody.

Patients with B cell malignancies have been the focus of the majority of CAR-T cell research, and preliminary findings are encouraging. In phase 1 trials, CAR-T cell treatment, for instance, showed full response rates of 69%–90% in young patients with relapsed or resistant acute lymphoblastic leukemia.

Lentiviral vectors have been effectively utilized in the context of gene therapy to modify hematopoietic stem cells, leading to the restoration of immunological function or the correction of hemoglobin abnormalities, to name only a few outcomes. For the treatment of B cell leukemias and lymphomas, chimeric antigen receptor (CAR) T cells have been highly effective, and this strategy mainly relies on lentivirus-mediated gene transfer. Here, we outline a typical procedure for producing lentivirus, ultra centrifuging it, and measuring the virus titer. The resultant virus can subsequently be employed in gene transfer tests in the lab, such as the development of CAR T cells.

Design of CAR-T Cells

 CARs are recombinant molecules made up of the variable antigen-binding regions of a monoclonal antibody. These CARs are connected to the T cell receptor complex's activation and Co-stimulatory domains. They are made up of an extracellular single-chain variable fragment (scFv) from an antibody that is connected to a transmembrane domain, a hinge peptide, and the intracellular T cell signaling domains of the T cell receptor.

Lentiviral Vectors: Transduction for CAR-T Cells

Viruses are the most often used vectors for gene therapy in both fundamental science and clinical research because of their high transfer efficiency, quick time reaching the clinically required numbers of grown T cells and accessibility of several viruses with various expression properties.

Lentivirus which has been genetically altered is the most widely used method of gene delivery. The ability of lentivirus-derived vectors to continue infecting non-dividing cells increases their capacity to transduce a range of cells, including quiescent and challenging-to-transduce cells.

Procedure 

◆For the manufacture of CAR-T cells, the viral vector is regarded as the primary raw material used in the transduction of CAR into T cells.

◆For each patient, a unique CAR-T cell product must be created, although the vector used to encode the CAR may be produced in vast quantities and kept at -80°C for four years.

◆At this temperature, frozen viral vectors remain stable for around 9 years. Because the ultimate result (a CAR-T cell) cannot be sterilized by filtration or any other sterility method, the vector that will be employed must be sterile.

◆Safety is improved by using a third-generation minimum lentiviral vector. For the batch production of viral vectors for cellular treatments, at least two weeks are needed. Growing sufficient HEK293T cells takes the majority of the time to manufacture substantial amounts of a replication-defective viral vector.

◆Plasmids are subsequently transfected into the newly formed cells, which results in the production of the minimum lentiviral vector. 

Typically, these plasmids are as follows:

1.A packaging design is known as Gag/Pol packaging, which encodes the viral structural protein (Gag) and enzyme (Pol).

2.A construct from a heterologous source that encodes the proper envelope glycoprotein and leads to the pseudo typing of a vector particle like VSV-G.

3.An auxiliary viral construct that expresses Rev; furthermore;

4.A plasmid vector carrying the CAR construct as well as other sequences required for RNA production, integration, and ERT packaging.

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