Gene transfer

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Creation of a CAR-T cell requires the ability to add genes of our choosing into T cells. As some viruses have evolved the ability to cross the cell membrane into cells and integrate their own genes into our genome, they can be modified and then employed to insert new genes into the selected target cells (Figure 1.3). These viruses are known as retroviruses, of which HIV is perhaps the most famous example. This is a subtype of retrovirus known as a lentivirus; both retroviruses and lentiviruses are used in gene transfer technologies.

Figure 1.3 Retroviral gene transfer. (1) Gene-encoding RNA enters the T cell in a modified lentivirus vector, where it is (2) reverse transcribed into DNA and (3) integrated into the T cell genome. (4) This ‘new’ DNA is transcribed into messenger RNA (mRNA), which then (5) directs the synthesis of a functional protein that enables (6) the T cell to express the antigen-specific chimeric antigen receptor (CAR). LTR, long terminal repeat; scFv, single-chain variable fragment.

Early on in CAR-T cell development, gene transfer was possible but difficult and very inefficient. As TCRs are made up of two separate proteins (α and β; see Figure 1.2b) encoded by two different genes, the process was even more difficult. This problem was made easier by designing a new, single, artificial gene that produced a functional protein that could both recognise antigen and transmit signals to the cell. This was achieved by connecting the previously separate heavy- and light-chain variable genes into one gene connected by a linker. These constructs are now termed first-generation CAR-T cells (Figure 1.4). 

Figure 1.4 In the first-generation CAR-T cells, the previously separate heavy- and light-chain variable domains were linked together with a flexible yet sturdy bridge known as a linker, creating a single-chain variable fragment (scFv). The scFv was then connected to a CD3ζ chain inside the cell by another flexible linking region called a hinge and a transmembrane domain that spanned the membrane. Second- and third-generation CAR-T cells also incorporate one or two intracellular co-stimulatory molecules (CD28, 4-1BB), respectively.