The progress in various approaches to gene therapy over the past decade has succeeded in reducing cost and increasing reliability. This has reached the point at which researchers can afford, from the point of view of both time and funding, to begin to combine gene therapies with other areas of medicine under development. In particular, reliable gene therapies targeting the controlling switches and dials of cell growth and regeneration should be a way to greatly improve the effectiveness of cell therapies and other, similar forms of regenerative medicine. The research reported below is a good example of the type, in which scientists combine a scaffold-based cell therapy with gene therapy to encourage local cells towards increased, controlled bone regrowth to replace severe fracture damage.
There are many methods of delivery for the introduction of therapeutic genes. The familiar use of viruses as a vector for the transfection of genes into a cell is just one class of approach – perhaps the most obvious one, given that viruses are in essence machines whose primary purpose is to place DNA into cells. But there are other approaches. Considered in the larger context, this diversity is a good thing, as greater competition and exploration always leads to a superior end result once all is said and done. The method used here is one of the pore-forming variations, in which one or another form of stimulus induces cells to open pores in the cell membrane and let in the DNA-bearing particles. In this case, the stimulus is physical, provided by cavitation of ultrasound-created microbubbles. It is worth bearing in mind that all of these methods have the potential to damage and kill cells, some more than others, and thus must be carefully calibrated. When it works, however, the results can be fairly impressive.
Investigators have successfully repaired severe limb fractures in laboratory animals with an innovative technique that cues bone to regrow its own tissue. If found to be safe and effective in humans, the pioneering method of combining ultrasound, stem cell and gene therapies could eventually replace grafting as a way to mend severely broken bones. “We are just at the beginning of a revolution in orthopedics. We’re combining an engineering approach with a biological approach to advance regenerative engineering, which we believe is the future of medicine.”
The new technique could provide a much-needed alternative to bone grafts. In their experiment, the investigators constructed a matrix of collagen, a protein the body uses to build bones, and implanted it in the gap between the two sides of a fractured leg bone in laboratory animals. This matrix recruited the fractured leg’s stem cells into the gap over two weeks. To initiate the bone repair process, the team delivered a bone-inducing gene directly into the stem cells, using an ultrasound pulse and microbubbles that facilitated the entry of the gene into the cells. Eight weeks after the surgery, the bone gap was closed and the leg fracture was healed in all the laboratory animals that received the treatment. Tests showed that the bone grown in the gap was as strong as that produced by surgical bone grafts.
We hypothesized that localized ultrasound-mediated, microbubble-enhanced therapeutic gene delivery to endogenous stem cells would induce efficient bone regeneration and fracture repair. To test this hypothesis, we surgically created a critical-sized bone fracture in the tibiae of Yucatán mini-pigs, a clinically relevant large animal model. A collagen scaffold was implanted in the fracture to facilitate recruitment of endogenous mesenchymal stem/progenitor cells (MSCs) into the fracture site. Two weeks later, transcutaneous ultrasound-mediated reporter gene delivery successfully transfected 40% of cells at the fracture site, and flow cytometry showed that 80% of the transfected cells expressed MSC markers. Human bone morphogenetic protein-6 (BMP-6) plasmid DNA was delivered using ultrasound in the same animal model, leading to transient expression and secretion of BMP-6 localized to the fracture area.
Micro-computed tomography and biomechanical analyses showed that ultrasound-mediated BMP-6 gene delivery led to complete radiographic and functional fracture healing in all animals 6 weeks after treatment, whereas nonunion was evident in control animals. Collectively, these findings demonstrate that ultrasound-mediated gene delivery to endogenous mesenchymal progenitor cells can effectively treat nonhealing bone fractures in large animals, thereby addressing a major orthopedic unmet need and offering new possibilities for clinical translation.