We propose to develop a system of in vivo ultrasound-mediated nucleic acid targeting for regenerative
medicine. As experts in skeletal tissue regeneration, we intend to provide proof of concept in orthopedic injury
models, first. Nonunion bone fractures and tendon/ligament tears are unmet clinical needs in the field of
orthopedic medicine and require the development of novel therapies. Current treatments to nonunion fractures
and ligament tears include autografts and allografts, which all involve serious complications and prolonged
periods of healing and loss of mobility. Bone Morphogenetic Protein (BMP) gene delivery, accomplished using
viruses, has been shown to induce healing of nonunion fractures in rodents and large animals. While viral
vectors are the most efficient gene delivery tools, they also introduce potential risks of tumorigenic and
immunogenic reactions. Nonviral vectors are considered safer for human use, albeit much less efficient for
gene expression. An alternative physical method of gene transfection termed sonoporation is especially
attractive for clinical applications due to the widespread use of ultrasound in the clinic today. Here, we will
employ local injection of microbubbles and subsequent ultrasound imaging and therapy to achieve high
efficiency local transfection. Recently we were able to demonstrate that ultrasound-based BMP-6 gene
delivery yielded complete segmental bone defect repair in a mini-pig model (Science Translational Medicine,
2017). Furthermore, a similar approach was used to deliver plasmid DNA to bone tunnels created for the
ligament reconstruction in a minipigs knee joints. However, currently three main hurdles prevent widespread
implementation of sonoporation for orthopaedic surgery: 1. The segmental fracture site poses a unique metal-
bone-soft tissue interface, which causes strong ultrasound reflections; 2. It is difficult to target the ultrasonic
pulse into narrow bone tunnels for tendon/ligament graft osseointegration, and 3. The control of cavitation
activity is also a hurdle and an opportunity for innovation and clinical advancements. Here we propose to rely
on the extensive expertise of the Ferrara group in the field of ultrasound–controlled drug delivery to overcome
the abovementioned hurdles and promote ultrasound mediated skeletal regenerative engineering to the clinic.
We propose two aims: Aim 1: Engineer and test image-guided transfection for critical-size bone fractures.
Goal: 1A) Develop transducer and software to sweep the US beam within a 3D volume while mapping and
controlling cavitation activity. Goal: 1B) Test the efficacy of fracture healing in a pig critical-size fracture model.
Aim 2: Engineer and test image-guided transfection customized for ligament/tendon bone tunnels. Goal: 2A)
Develop transducer and software to direct the US beam within a bone tunnel while mapping and controlling
cavitation activity. 2B) Test the efficacy of enhanced repair in a pig ligament reconstruction model. If
successful, this project could be highly beneficial for numerous clinical applications beyond orthopedic targets,
including cancer therapy.