Engineering T cells using VDJ targeting:

VDJ targeting integrates CAR or TCR genes into the endogenous TCR loci, independent of promiscuously integrating vectors and exogenous nucleases. Thus, it allows natively controlled expression from the strong endogenous promoter in highly potent naïve cells, and it further allows subsequent activation and affinity maturation for effective immune response, memory retention and diminished antigenic escape. VDJ targeting employs rAAV as well as the natural process of V(D)J recombination, taking place in developing lymphocytes. Natural V(D)J recombination is catalyzed by the RAG complex. RAG recognizes recombination signal sequences (RSSs) and recombines them to join together appropriate gene segments. Flanking of a receptor/Ab gene with appropriate RSSs will allow its recognition as a substrate of RAG-mediated targeted insertion into the endogenous TCR/Ab chain locus, but only if the vector transduces appropriate lymphocyte precursors, in which RAG is active. With VDJ targeting, immunotherapy will be more safe and more potent, and could finally be scaled up from the isolated specialized centers to the community clinic, and from benefitting selected patients to treating the large population in need.