Engineering BV to efficiently deliver large genetic payloads

Summary: Efficient in vivo delivery of large DNA constructs encoding RNA and proteins will provide a powerful tool for a wide range of applications in health, including the use of genome engineering for understanding and controlling biological functions, and gene therapies for treating human disease. Existing in vivo delivery vehicles, such as adeno-associated airal vectors (AAVs) and lipid nanoparticles (LNPs) lack the ability to deliver large DNA constructs in vivo. Baculoviral (BV) vectors offer a potential solution to this unmet need. Derived from an insect virus, BV vectors have an extraordinary DNA payload packing capacity (up to 300 kb), can infect both dividing and nondividing cells, only replicate in insect cells thus are considered safe and nonpathogenic to humans. However, there are major challenges for BV to become a vehicle for in vivo gene delivery. BVs delivered systemically in vivo are often inactivated by the innate complement system, hindering their ability to transduce cells efficiently in vivo. BVs also have weak interactions with mammalian cell surface proteins, and those internalized can be trapped in the endosomes, further reducing transduction efficiency. This proposed study aims to engineer BV vectors for efficient in vivo delivery of large DNA constructs, by screening diverse BV surface- displayed factors including cell adhesion, endosomal escape and complement protection factors. We hypothesize that, thorough the expression of an optimal set of BV surface-displayed factors, BVs can be programmed to deliver large therapeutic DNA payloads in vivo with high efficiency. This hypothesis is based on our preliminary data demonstrating that in vivo delivery efficiency of engineered BV vectors can be synergistically enhanced by co-expression of endosomal escape and complement protection factors. In Aim 1 studies we will carry out high-throughput screening to identify combinations of cell adhesion and endosomal escape factors that improve the transduction efficiency of BVs. In Aim 2 studies we will identify complement protection factors and combine them with the optimal cell adhesion and endosomal escape factors, and determine if the in vivo delivery efficiency of large therapeutic DNA cargos can be enhanced significantly. Successfully completion of this work will result in gene delivery vectors capable of efficiently expressing large DNA payloads in vivo, enabling safter and more effective therapeutic strategies for a wider array of diseases.