VDJ Targeting – Nuclease-Free B Cells Engineering Using VDJ Recombination

B cell engineering has remarkably evolved is the last few years, harnessing the potential of B cells to produce therapeutic antibodies and to provide long-term protein-replacement.
Most current B cell engineering approaches use CRISPR-Cas9 nucleases to target the integration of an antibody coding gene into the immunoglobulin heavy locus of mature B cells. However, engineering of mature B cells requires pre-activation leading to the formation of DSBs in the process of class switch recombination (CSR) in addition to the CRISPR/Cas9 breaks. While the erroneous repair of such CSR induced DSBs might lead in rare cases to chromosomal translocations and the formation of B cell lymphomas, the introduction of an additional DSB using CRISPR-Cas9 may severely aggravates such genotoxic risk.
Here we describe the engineering of primary developing B cells using a novel nuclease-free approach called VDJ targeting. A recombinant adeno-associated viral vector (rAVV), coding for an Ab gene, is inserted into the IgH locus by the endogenous recombination activating gene (RAG) complex during V(D)J recombination, without use of exogenous nucleases. Here we demonstrate for the first time, not only high transduction rates (>40%) of actively developing mouse B cells using AAV-DJ, but also the integration and expression of a broadly neutralizing antibody against HIV-1.
In addition, we successfully devised an in-vitro differentiation protocol of the precursor B cells into immature and mature B cells. Up to 43% out the CD19+ population displayed an immature phenotype and an additional 40% displayed a mature phenotype. These cells will be further capable of undergoing activation and differentiation into memory and plasma cells in future experiments.