Sonoporation Increases Therapeutic Efficacy of Inducible and Constitutive BMP2/7 In Vivo Gene Delivery

Abstract

An ideal novel treatment for bone defects should provide regeneration without autologous or allogenous grafting, exogenous cells, growth factors, or biomaterials while ensuring spatial and temporal control as well as safety. Therefore, a novel osteoinductive nonviral in vivo gene therapy approach using sonoporation was investigated in ectopic and orthotopic models. Constitutive or regulated, doxycycline-inducible, bone morphogenetic protein 2 and 7 coexpression plasmids were repeatedly applied for 5 days. Ectopic and orthotopic gene transfer efficacy was monitored by coapplication of a luciferase plasmid and bioluminescence imaging. Orthotopic plasmid DNA distribution was investigated using a novel plasmid-labeling method. Luciferase imaging demonstrated an increased trend (61% vs. 100%) of gene transfer efficacy, and micro-computed tomography evaluation showed significantly enhanced frequency of ectopic bone formation for sonoporation compared with passive gene delivery (46% vs. 100%) dependent on applied ultrasound power. Bone formation by the inducible system (83%) was stringently controlled by doxycycline in vivo, and no ectopic bone formation was observed without induction or with passive gene transfer without sonoporation. Orthotopic evaluation in a rat femur segmental defect model demonstrated an increased trend of gene transfer efficacy using sonoporation. Investigation of DNA distribution demonstrated extensive binding of plasmid DNA to bone tissue. Sonoporated animals displayed a potentially increased union rate (33%) without extensive callus formation or heterotopic ossification. We conclude that sonoporation of BMP2/7 coexpression plasmids is a feasible, minimally invasive method for osteoinduction and that improvement of bone regeneration by sonoporative gene delivery is superior to passive gene delivery.

Evaluation of fibrin-based gene-activated matrices for BMP2/7 plasmid codelivery in a rat nonunion model

“…Results of this study highlight the importance of promoting non-viral gene transfer by alternative methods. Currently sonoporation or electroporation seem to be the most promising strategies in terms of safety, efficacy and minimal invasiveness [9,[29][30][31]. Further studies are needed to clarify if those strategies are capable to increase bone growth in a clinical relevant segmental bone defect model.…”

“…Application of BMP2 and BMP7 plasmids in fibrin GAMs without cationic polymer lead to variable results in bone volume without sufficient new bone formation to bridge the femoral critical size defect. It indicates a potential advantage of delivering BMP2 and 7 transgenes in fibrin gels for tissue regeneration if enhanced by methods such as the recently developed matrix-assisted sonoporation [33] or methods without application of biomaterials relying only on physical stimuli for gene delivery such as sonoporation or electroporation [9,31]. Black and grey areas represent unions and narrow unions respectively.…”

In situ bone tissue engineering via ultrasound-mediated gene delivery to endogenous progenitor cells in mini-pigs

“…Feichtinger et al . (25) used microbubble-enhanced, ultrasound-mediated gene delivery of BMP-2 and BMP-7 in a femur nonunion model in rats. Despite successfully inducing gene overexpression and ectopic bone formation using repetitive treatments, they could not induce significant orthotopic bone formation in a fracture site.…”

“…Although ultrasound-mediated BMP gene delivery was shown to induce ectopic bone formation in vivo (25–27), previous attempts at fracture repair were not successful (25). Here, we hypothesized that microbubble-enhanced, ultrasound-mediated gene delivery of a BMP gene would lead to efficient bone regeneration and fracture healing in a clinically relevant, large animal model (porcine).…”

Healing of massive segmental femoral bone defects in minipigs by allogenic ASCs engineered with FLPo/Frt-based baculovirus vectors

“…Among the numerous bone tissue engineering approaches, gene therapy offers a promising alternative for sustained release of osteoinductive and/or angiogenic factors, and may be achieved by direct in vivo delivery [29] or ex vivo delivery combined with cell therapy [6]. The majority of these studies have used small animals such as mice, rats or rabbits (for review see Refs.…”