Engineering B cells using Switch targeting

We plan to greatly enhance the safety, efficacy and the scalability of our approach by engineering the B cells without CRISPR/Cas9. CRISPR/Cas9 cleavage is associated with both on-target and off-target mutagenesis as well as with gross chromosomal aberrations. Importantly, the activation of B cells is associated also with natural double strand breaks (DSB), initiated by activation-induced-cytidine-deaminase (AID) and leading to CSR. Therefore, applying CRISPR/Cas9 to activated B cells may bare a combined risk for potentially oncogenic translocations. To avoid these risks, we reasoned that bNAb genes could be targeted to AID induced breaks at the IgH locus using adeno associated viral vectors (AAV) without CRISPR/Cas9. Even a low integration efficiency may suffice, as engineered B cells could readily be selected or sorted and then propagated to therapeutically relevant numbers before adoptive transfer. We plan to establish B cell engineering without nucleases as a potentially one-shot treatment for infections by HIV and other pathogens. We further plan to harness nuclease-free B cell engineering as a constitutive or inducible protein replacement therapy.