Development of an MFG-Based Retroviral Vector System for Secretion of High Levels of Functionally Active Human BMP4

Abstract

We sought to develop a retroviral vector system that would produce secretion of high levels of bone morphogenetic protein (BMP)-4 by optimizing the expression construct and developing an improved retroviral vector. Replacement of the propeptide domain of BMP4 with that of BMP2 increased the secretion level of mature BMP4 protein in transduced cells. The intact BMP2 pro-peptide sequence was essential, as deletion of a small part of the propeptide sequence of BMP2 from the BMP2/4 hybrid construct diminished BMP4 expression and secretion. Addition of a hemaglutinin tag to the carboxy terminus of BMP4 abolished the bioactivity of secreted BMP4. Transduction of rat marrow stromal cells (and fibroblasts) with an MFG-based retroviral vector pseudotyped with VSV-G envelope containing this BMP2/4 hybrid expression construct led to secretion of very high levels of mature BMP4 in conditioned medium (up to 1 microg/10(6) cells/24 hours). The secreted BMP4 was biologically active, as it induced alkaline phosphatase expression in C2C12 cells. The transduced rat marrow stromal cells expressing mature BMP4 induced de novo ectopic bone formation in syngenic immune-competent rats. We have developed an MFG-based retroviral vector system that causes secretion of high levels of functionally active human BMP4 protein.

Ex vivo gene therapy with stromal cells transduced with a retroviral vector containing the BMP4 gene completely heals critical size calvarial defect in rats

“…We have been able to achieve these requirements with an MLV-based retroviral vector that has the gene for the mature human BMP4 protein coupled to a secretion signal from BMP2. 17 In the present study, we verified the production of BMP by immunostaining and on Western blots using specific BMP4 antibodies in stromal cells transduced with BMP4 or control MLV virus. A high level of BMP4 transduced cells (>60%) stained positive for the transgene (Figure 1a), while cells transduced with a control LacZ vector show only background staining (Figure 1b).…”

“…In this regard, our recently developed MLV-based vector system appeared to overcome these limitations. 17 Accordingly, we reported that the use of the BMP2/4 hybrid expression construct, in which the propeptide domain of BMP4 was replaced by the corresponding propeptide domain of BMP2, greatly enhanced the secretion of mature and biologically active BMP4 by the transduced cells. 17 The titer of our MLV-based BMP4 retroviral vector was in the range of 1 × 10 6 to 5 × 10 6 transduction units per ml.…”

“…17 Accordingly, we reported that the use of the BMP2/4 hybrid expression construct, in which the propeptide domain of BMP4 was replaced by the corresponding propeptide domain of BMP2, greatly enhanced the secretion of mature and biologically active BMP4 by the transduced cells. 17 The titer of our MLV-based BMP4 retroviral vector was in the range of 1 × 10 6 to 5 × 10 6 transduction units per ml. 17 Although it is relatively low compared with adenoviral vectors, it is high compared with most available retroviral vector systems.…”

“…17 The titer of our MLV-based BMP4 retroviral vector was in the range of 1 × 10 6 to 5 × 10 6 transduction units per ml. 17 Although it is relatively low compared with adenoviral vectors, it is high compared with most available retroviral vector systems. Consequently, our recent advance in the development of a high titer MLV-based BMP4 retroviral vector that allows secretion of high levels of functionally active human BMP4 in transduced marrow stromal cells 17 proGene Therapy vides us with an important tool to test the feasibility of development of an MLV-based retroviral vector system for gene therapy of bone repair and regeneration.…”

“…In contrast, retroviral vector systems do not elicit substantial immune responses and allow stable integration of the transgene into the host genome and, therefore, could have important advantages over adenoviral vectors. In this regard, we have recently developed an MLV-based retroviral vector system that allows the secretion of biologically active BMP4 protein from transduced marrow stromal cells in amounts often exceeding 1 g/10 6 cells/24 h. 17 Consequently, we sought in this study to use our recently developed MLV-based BMP4 vector system to assess the usefulness of the retroviral vector-based system for gene therapy of bone repair and regeneration. In support of the contention that MLVbased vectors can be an effective means to deliver a bone growing gene for gene therapy of bone repair and regeneration, several recent reports have obtained promising results with MLV-based vectors in gene therapy of bone defects.…”