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Determination of effective rAAV-mediated gene transfer conditions to support chondrogenic differentiation processes in human primary bone marrow aspirates

Gene Therapy volume 22, pages 50–57 (2015)Cite this article

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Abstract

The genetic modification of freshly aspirated bone marrow may provide convenient tools to enhance the regenerative capacities of cartilage defects compared with the complex manipulation of isolated progenitor cells. In the present study, we examined the ability and safety of recombinant adeno-associated virus (rAAV) serotype 2 vectors to deliver various reporter gene sequences in primary human bone marrow aspirates over time without altering the chondrogenic processes in the samples. The results demonstrate that successful rAAV-mediated gene transfer and expression of the lacZ and red fluorescent protein marker genes were achieved in transduced aspirates at very high efficiencies (90–94%) and over extended periods of time (up to 125 days) upon treatment with hirudin, an alternative anticoagulant that does not prevent the adsorption of the rAAV-2 particles at the surface of their targets compared with heparin. Application of rAAV was safe, displaying neither cytotoxic nor detrimental effects on the cellular and proliferative activities or on the chondrogenic processes in the aspirates especially using an optimal dose of 0.5 mg ml−1 hirudin, and application of the potent SOX9 transcription factor even enhanced these processes while counteracting hypertrophic differentiation. The current findings demonstrate the clinical value of this class of vector to durably and safely modify bone marrow aspirates as a means to further develop convenient therapeutic approaches to improve the healing of cartilage defects.

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Acknowledgements

The authors thank RJ Samulski (The Gene Therapy Center, University of North Carolina, Chapel Hill, NC, USA) and X Xiao (The Gene Therapy Center, University of Pittsburgh, Pittsburgh, PA, USA) for providing genomic AAV-2 plasmid clones and the 293 cell line. This work was supported by grants from The German Research Society (DFG) and The AO Foundation, Davos, Switzerland.

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Authors and Affiliations

  1. Center of Experimental Orthopaedics, Saarland University Medical Center, Homburg/Saar, Germany

    A Rey-Rico, J Frisch, J K Venkatesan, G Schmitt, H Madry & M Cucchiarini

  2. Department of Orthopaedic Surgery, Saarland University Medical Center, Homburg/Saar, Germany

    H Madry

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  1. A Rey-Rico
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  2. J Frisch
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Correspondence to M Cucchiarini.

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Rey-Rico, A., Frisch, J., Venkatesan, J. et al. Determination of effective rAAV-mediated gene transfer conditions to support chondrogenic differentiation processes in human primary bone marrow aspirates. Gene Ther 22, 50–57 (2015). https://doi.org/10.1038/gt.2014.90

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