Local immunomodulation with CD4 and CD8 antibodies, but not cyclosporine A, improves osteogenesis induced by ADhBMP9 gene therapy

Li, J Z; Li, H; Hankins, Gerald R.; Dunford, Bradley; Helm, Gregory A.

doi:10.1038/sj.gt.3302502

Cited by 8 publications

(4 citation statements)

References 41 publications

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“…In addition to BMPs and the Runx2 transcription factor, promising results have been achieved with strategies focusing on VEGF and RANK-L, 53 constitutively active BMP receptors, 52 LIM-mineralization protein, 54 synergistic approaches incorporating multiple transgenes 46,55 and transgenes supplemented with glucocorticoids 56 or immunosuppressive agents. 57,58 Efficacy of these approaches may also be dependent on cell type 38 and in vitro preculture of cell-scaffold composites. 19 Nevertheless, inducible expression systems show significant promise in developing safe and controlled gene therapy strategies for bone tissue engineering.…”

Section: Discussionmentioning

confidence: 99%

Inducible regulation of Runx2-stimulated osteogenesis

et al. 2006

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Ex vivo gene therapy is a promising approach to orthopedic regenerative medicine. These strategies typically focus on the constitutive overexpression of osteogenic factors to induce osteoblastic differentiation and matrix mineralization. However, the unregulated production of osteoinductive molecules has also resulted in abnormal bone formation and tumorigenesis. To address these limitations, this work describes a retroviral system to deliver the Runx2 osteoblastic transcription factor under control of the tetracycline-inducible (tet-off) promoter in primary skeletal myoblasts. Runx2 expression was tightly regulated by anhydrotetracyline (aTc) concentration in cell culture media. Osteoblastic gene expression, alkaline phosphatase activity, and matrix mineralization were also controlled by aTc in a dose-dependent manner. Additionally, osteoblastic differentiation was temporally regulated by adding and removing aTc from the culture media. Engineered cells were seeded onto collagen scaffolds and implanted intramuscularly in the hind limbs of syngeneic mice. In vivo mineralization by these constructs was regulated by supplementing the drinking water with aTc, as demonstrated by micro-computed tomography and histological analyses. Collectively, these results present a novel system for regulating osteoblastic differentiation of a clinically relevant autologous cell source. This system is significant to developing controlled and effective orthopedic gene therapy strategies and studying the regulation of osteoblastic differentiation.

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Section: Discussionmentioning

confidence: 99%

Inducible regulation of Runx2-stimulated osteogenesis

et al. 2006

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“…Promising results have been achieved by focusing on other members of the BMP family [Krebsbach et al, 2000; Jane et al, 2002], VEGF and RANK‐L [Ito et al, 2005], constitutively active BMP receptors [Koefoed et al, 2005], and LIM‐mineralization proteins [Pola et al, 2004]. Other studies have demonstrated synergistic effects by incorporating multiple transgenes [Yang et al, 2003; Zhao et al, 2005a] and transgenes supplemented with glucocorticoids [Phillips et al, 2006a] or immunosuppressive agents [Kaihara et al, 2004; Li et al, 2005]. Additionally, the limitations of uncontrolled growth factor secretion and constitutive transgene expression are also being addressed with tissue‐specific promoters [Hou et al, 1999], inducible expression systems [Moutsatsos et al, 2001; Gafni et al, 2004; Peng et al, 2004; Gersbach et al, 2006], and co‐expression of regulatory factors [Peng et al, 2005].…”

Section: Discussionmentioning

confidence: 99%

In vitro and in vivo osteoblastic differentiation of BMP‐2‐ and Runx2‐engineered skeletal myoblasts

Gersbach

Guldberg

Garcı́a

2006

J of Cellular Biochemistry

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Genetic engineering with osteogenic factors is a promising approach for cell-based therapeutics and orthopedic regeneration. However, the relative efficacy of different strategies for inducing osteoblastic differentiation remains unclear and is further complicated by varied delivery vehicles, cell types, and evaluation criteria. In order to elucidate the effects of distinct gene-based strategies, we quantitatively evaluated osteoblastic differentiation and mineralization of primary skeletal myoblasts overexpressing either the BMP-2 growth factor or Runx2 transcription factor. Retroviral delivery of BMP-2 or Runx2 stimulated differentiation into an osteoblastic phenotype, as demonstrated by the induction of osteogenic gene expression, alkaline phosphatase activity, and matrix mineralization in monolayer culture and on collagen scaffolds both in vitro and in an intramuscular site in vivo. In general, BMP-2 stimulated osteoblastic markers faster and to a greater extent than Runx2, although we also identified experimental conditions under which these two factors produced similar effects. Additionally, Runx2-engineered cells did not utilize paracrine signaling via secreted osteogenic factors, in contrast to cells overexpressing BMP-2, as demonstrated by conditioned media studies and activation of Smad signaling. These results emphasize the complexity of gene therapy-based orthopedic therapeutics as an integrated relationship of differentiation state, construct maturation, and paracrine signaling of osteogenic cells. This study is significant in evaluating proposed therapeutic systems and defining a successful strategy for integrating gene medicine and orthopedic regeneration.

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“…After antibody staining, the labeled white blood cells were analyzed by performing flow cytometry. 33,35 The percentages of single-stained and double-stained cells were calculated based on the total number of white blood cells.…”

Section: Measuring Levels Of Immune Cells In Rat Bloodmentioning

confidence: 99%

Different Osteogenic Potentials of Recombinant Human BMP-6 Adeno-Associated Virus and Adenovirus in Two Rat Strains

Li²,

Hankins³

et al. 2006

Tissue Engineering

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The osteogenic potential of AAV5hBMP6 was compared with that of ADhBMP6 in immunodeficient and immunocompetent rats. AAV5hBMP6 (2.3 x 10(12) particles) and ADhBMP6 (5 x 10(7) PFU) elicited viral antibody production in immunocompetent rats. Among rats that received AAV5hBMP6, the earliest time points at which the bone was visible under CT scanner were 30 days in 2-month-old Sprague-Dawley (SD) rats and 60 days in 18-month-old SD rats. The mean volumes of ectopic bone 90 days after viral injection were 0.31 +/- 0.14 cm(3) in athymic nude rats, 0.64 +/- 0.12 cm(3) in 2-month-old SD rats, and 0.21 +/- 0.10 cm(3) in 18-month-old SD rats. In contrast, among rats that received ADhBMP6, the earliest time points to observe the bone formation by CT scan were 15 days in 2-month-old rats and no bone formation in 18-month-old SD rats. The mean volumes of ectopic bone were 4.17 +/- 0.05 cm(3) in athymic nude rats and 0.06 +/- 0.03 cm(3) in 2-month-old SD rats. Although both types of viruses induced an immune response in immunocompetent animals, this response played different roles in the process of bone formation induced by the BMP6 vectors.

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Local immunomodulation with CD4 and CD8 antibodies, but not cyclosporine A, improves osteogenesis induced by ADhBMP9 gene therapy

Cited by 8 publications

References 41 publications

Inducible regulation of Runx2-stimulated osteogenesis

Inducible regulation of Runx2-stimulated osteogenesis

In vitro and in vivo osteoblastic differentiation of BMP‐2‐ and Runx2‐engineered skeletal myoblasts

Different Osteogenic Potentials of Recombinant Human BMP-6 Adeno-Associated Virus and Adenovirus in Two Rat Strains

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