Lentivirus-mediated gene transfer induces long-term transgene expression of BMP-2 in vitro and new bone formation in vivo

Abstract

We examined the potential of ex vivo gene therapy to enhance bone repair using lentiviral vectors encoding either enhanced green fluorescent protein (EGFP) as a reporter gene or bone morphogenetic protein-2 (BMP-2) downstream of either the cytomegalovirus immediate early (CMV) promoter or the murine leukemia virus long terminal repeat (RhMLV) promoter derived from a murine retrovirus adapted to replicate in a rhesus macaque. In vitro, rat bone marrow stromal cells (BMSCs) transduced with Lenti-CMV-EGFP or Lenti-RhMLV-EGFP demonstrated over 90% transduction efficiency at 1 week and continued to demonstrate stable expression for 8 weeks. ELISA results demonstrated that lentivirus-mediated gene transfer into BMSCs induced stable BMP-2 production in vitro for 8 weeks. Increased EGFP and BMP-2 production was noted with the RhMLV promoter. In addition, we implanted BMSCs transduced with Lenti-RhMLV-BMP-2 into a muscle pouch in the hind limbs of severe combined immune deficient mice. Robust bone formation was noted in animals that received Lenti-RhMLV-BMP-2 cells at 3 weeks. These results demonstrate that lentiviral vectors expressing BMP-2 can induce long-term gene expression in vitro and new bone formation in vivo under the control of the RhMLV promoter. Prolonged gene expression may be advantageous when developing tissue engineering strategies to repair large bone defects.

Lentiviral RNAi-induced downregulation of adenosine kinase in human mesenchymal stem cell grafts: A novel perspective for seizure control

“…(iii) Gene expression with lentiviral vectors can effectively be achieved in hMSCs (Totsugawa et al, 2002) and continuous stable gene expression was demonstrated throughout differentiation (Sugiyama et al, 2005). (iv) Functional RNA interference in hMSCs throughout differentiation is well documented (Hoelters et al, 2005).…”

Local gene transfer to calcified tissue cells using prolonged infusion of a lentiviral vector

“…Expression of matrix proteins, such as AMBN, offers the possibility to study their function and possible application in the development of novel strategies for bone regeneration. In addition, because lentiviruses can sustain expression of a transgene over periods of several months, 29,30,34,49 they can be advantageously exploited for rescue experiments in knockout mice with canonical and mutated proteins. …”

“…28 Efficient lentivirus-mediated gene transfer has been demonstrated in several cells and tissues, including skeletal muscle, 29 salivary glands, 30 liver, 29 neural tissues 31,32 and keratinocytes. 33 There are only few studies on the administration of lentiviral vectors (LVs) at calcified tissue sites and these dealt with the periosseous implantation of cells infected ex vivo 21,34 and local injection of the vector into the synovium. 10,35 Local delivery of viral vectors into bone remains problematic because it has so far been limited to readily accessible anatomical sites such as the synovial cavity and bone defects, and relatively large amounts of vector and its local persistence are required to successfully infect a sufficient number of cells.…”

Efficacy of minimally invasive techniques for enhancement of fracture healing: evidence today

“…Gene transfer could overcome these limitations. Several viral vectors carrying BMP-2 cDNA have been studied on animal models over the years [4,5,104]. All showed promising results for future clinical application.…”