Enhancing in vivo B cell gene delivery using targeted lentiviral vectors and transduction enhancers

B cells occupy a distinct niche in humoral immunity, and engineered B cells that leverage their unique biological functions hold significant promise for therapeutic applications. Here, we combine chemical and virological strategies to establish a method for in situ engineering of B cells by optimizing formulations of CD20-targeted Nipah paramyxovirus-pseudotyped lentiviral vectors (LVs) coupled with the use of transduction enhancers (TEs). Select TE combinations enabled greater than 3-fold improvements in transduction efficiency of isolated B cells as well as B cells within human PBMC populations in vitro, without compromising targeting specificity. In particular, a combination of poloxamer 407 and valproic acid, as well as a formulation containing four distinct TEs, significantly enhanced transduction of circulating human B cells in PBMC-engrafted NSG mice compared with no-TE controls. This enhancement resulted in more than a 20-fold increase in serum levels of a recombinant anti-RSV antibody by day 26, with no detectable safety concerns or off-target transduction. Together, these findings demonstrate that appropriate formulation of LVs with selected TEs enables effective and specific in vivo B-cell engineering, supporting the development of new B-cell-based therapeutic strategies that integrate gene and cell therapy approaches to reprogram immunity. STATEMENT OF SIGNIFICANCE: Efficient in vivo cell engineering remains limited by two major challenges: achieving cell-type specificity and attaining sufficient gene transfer efficiency for therapeutic relevance. This study demonstrates that formulation of targeted lentiviral vectors with well-characterized transduction enhancers can substantially improve in vivo gene delivery to B cells without compromising specificity. By combining CD20-targeted paramyxovirus-pseudotyped lentiviral vectors with selected small-molecule and polymer-based enhancers, we achieve markedly enhanced transduction of human B cells in vitro and in vivo. Given the intrinsic difficulty of transducing circulating B cells, these findings establish a broadly applicable strategy for improving targeted gene delivery through vector formulation.