ABSTRACT Both private entities and academic groups are pioneering B cell editing for therapeutic purposes, either to express therapeutic antibodies from their native Ig loci, or other transgenes from an ectopic locus. BCR editing is currently performed with CRISPR/Cas system and a homology- directed repair template. Non-BCR trasngenes are introduced by retroviral transduction or transposon-based random insertion. Both nuclease-based and insertion-based B cell engineering techniques carry risks associated with chromosomal deletion (CRISPR/Cas) or insertional mutagenesis (retroviruses/transposons). Furthermore, efficient editing is only possible by isolating and editing B cells ex vivo meaning that therapies based on these editing protocols will likely be very expensive. We have discovered a method of B cell editing that requires no exogenous nucleases and does not rely on random insertion. Our method relies on transducing class-switching B cells with a DNA template supplied by a recombinant adeno-associated virus (rAAV) vector. The inverted terminal repeat (ITR) sequences in the rAAV naturally integrate into double-strand breaks created by the B cell class-switch machinery. With the right expression cassette designs, we can replace the endogenous heavy chain variable (VH) segment or even express a non-antibody transgene from within the BCR locus. This nuclease-free technique has potential advantages in terms of safety and, because it requires only a single rAAV transduction event, it also promises a simple, cost effective means of editing B cells in vivo. Here we aim to develop our nuclease-free editing technique and provide proof-of-concept for therapeutic applications. In Aim 1, we will optimize the design of our rAAV-delivered repair template, and demonstrate the relative safety of our approach compared to CRISPR/Cas-based editing. In Aim 2, we test different expression cassette designs for antibody and non-antibody transgene expression and determine whether or not inclusion of cis-acting genetic elements that increase somatic hypermutation can enhance affinity maturation of our edited B cells. In Aim 3, we will address in vivo editing. We will determine whether or not in vivo nuclease-free editing efficiency can be enhanced by vaccinating mice prior to rAAV administration to drive B cell class switching and optimize our rAAV doses and timing relative to the pre-vaccination step. We will also demonstrate the ability of our edited B cell system to produce recombinant antibodies (BCR editing) and erythropoietin (non-antibody transgene expression) in mice.