Local administration of AAV-DJ pseudoserotype expressing COX2 provided early onset of transgene expression and promoted bone fracture healing in mice

Abstract

We have previously obtained compelling proof-of-principle evidence for COX2 gene therapy for fracture repair using integrating retroviral vectors. For this therapy to be suitable for patient uses, a suitable vector with high safety profile must be used. Accordingly, this study sought to evaluate the feasibility of AAV as the vector for this COX2 gene therapy, because AAV raises less safety issues than the retroviral vectors used previously. However, an appropriate AAV serotype is required to provide early increase in and adequate level of COX2 expression that is needed for fracture repair. Herein, we reported that AAV-DJ, an artificial AAV pseudoserotype, is highly effective in delivering COX2 gene to fracture sites in a mouse femoral fracture model. Compared with AAV-2, the use of AAV-DJ led to ~5-fold increase in infectivity in mesenchymal stem cells (MSCs) and provided an earlier and significantly higher level of transgene expression at the fracture site. Injection of this vector at a dose of 7.5 × 10(11) genomic copies led to high COX2 level at the fracture site on day 3 after injections and significantly promoted fracture union at 21 days, as analyzed by radiography and μ-CT. The therapeutic effect appears to involve enhanced osteoblastic differentiation of MSCs and remodeling of callus tissues to laminar bone. This interpretation is supported by the enhanced expression of several key genes participating in the fracture repair process. In conclusion, AAV-DJ is a promising serotype for the AAV-based COX2 gene therapy of fracture repair in humans.

1,25-Dihydroxyvitamin D suppresses M1 macrophages and promotes M2 differentiation at bone injury sites

“…Third, a deficiency of M1 macrophage-recruiting molecules (i.e., monocyte chemoattractant protein 1 receptor, also known as C-C motif chemokine receptor 2 [CCR-2]), led to compromised fracture healing (40). Fourth, cyclooxygenase 2 (COX-2) gene therapy accelerated fracture healing (41)(42)(43), and inhibitors of COX-2 compromised fracture healing primarily at the proinflammatory phase of fracture healing (44). This role of COX-2 in fracture repair is at least partially related to M1 macrophages because M1 macrophages depend on COX-2 to enhance in vitro osteoblast differentiation in MSCs (13).…”

Ultramicroscopy as a novel tool to unravel the tropism of AAV gene therapy vectors in the brain

“…The combination of non-pathogenicity and efficient persistence of AAVs makes it an attractive delivery vehicle for gene therapy applications24. To date, twelve naturally occurring human AAV serotypes25 and more than one hundred serotypes from non-human primates and other species have been discovered2627. The identification of novel serotypes, such as AAV9 and AAVrh10, directed under the action of various promoters, as well as the repertoire of engineered chimera vectors, are likely to improve and enlarge the AAV application range222829.…”

Differential Cellular Tropism of Lentivirus and Adeno-Associated Virus in the Brain of Cynomolgus Monkey

“…To identify which cells were infected with AAV, 25 µl AAV-GFP diluted with saline was injected into putamen of cynomolgus monkey unilaterally. As it is described already, AAV-DJ has been widely used for the vehicle of the gene delivery because it has a higher transduction efficiency [ 11 ]. Therefore, we packaged with AAV-DJ.…”

“…In that study, the authors found that AAV serotype 5 displayed the most efficient transduction of neurons in this brain region. Recently, AAV-DJ vector was generated as one of the recombinogenic hybrid vector from gene shuffling of eight serotypes and has been widely used across a broad range of cell types because it has a higher transduction efficiency both in vitro and in vivo [ 10 11 ]. Even though AAV serotype 5 displayed the most efficient transduction of neurons in the African green monkey brain region [ 9 ], AAV-DJ has not been tested in the primate.…”