RAAV-Mediated Overexpression of SOX9 and TGF-β via Carbon Dot-Guided Vector Delivery Enhances the Biological Activities in Human Bone Marrow-Derived Mesenchymal Stromal Cells.

Weikun Meng,Luc Lebeau,Françoise Pons,Susanne Speicher-Mentges,Jagadeesh K Venkatesan,Magali Cucchiarini,Gertrud Schmitt,Mickaël Claudel,Ana Rey-Rico

doi:10.3390/nano10050855

Abstract

Scaffold-assisted gene therapy is a highly promising tool to treat articular cartilage lesions upon direct delivery of chondrogenic candidate sequences. The goal of this study was to examine the feasibility and benefits of providing highly chondroreparative agents, the cartilage-specific sex-determining region Y-type high-mobility group 9 (SOX9) transcription factor or the transforming growth factor beta (TGF-β), to human bone marrow-derived mesenchymal stromal cells (hMSCs) via clinically adapted, independent recombinant adeno-associated virus (rAAV) vectors formulated with carbon dots (CDs), a novel class of carbon-dominated nanomaterials. Effective complexation and release of a reporter rAAV-lacZ vector was achieved using four different CDs elaborated from 1-citric acid and pentaethylenehexamine (CD-1); 2-citric acid, poly(ethylene glycol) monomethyl ether (MW 550 Da), and N,N-dimethylethylenediamine (CD-2); 3-citric acid, branched poly(ethylenimine) (MW 600 Da), and poly(ethylene glycol) monomethyl ether (MW 2 kDa) (CD-3); and 4-citric acid and branched poly(ethylenimine) (MW 600 Da) (CD-4), allowing for the genetic modification of hMSCs. Among the nanoparticles, CD-2 showed an optimal ability for rAAV delivery (up to 2.2-fold increase in lacZ expression relative to free vector treatment with 100% cell viability for at least 10 days, the longest time point examined). Administration of therapeutic (SOX9, TGF-β) rAAV vectors in hMSCs via CD-2 led to the effective overexpression of each independent transgene, promoting enhanced cell proliferation (TGF-β) and cartilage matrix deposition (glycosaminoglycans, type-II collagen) for at least 21 days relative to control treatments (CD-2 lacking rAAV or associated to rAAV-lacZ), while advantageously restricting undesirable type-I and -X collagen deposition. These results reveal the potential of CD-guided rAAV gene administration in hMSCs as safe, non-invasive systems for translational strategies to enhance cartilage repair.

Highlights

Articular cartilage lesions represent serious clinical issues in orthopaedics as this specialized tissue does not fully heal on itself by lack of vascularization and of local chondroregenerative cells that may repopulate the defects [1,2]
It remains to be seen whether carbon dots (CDs) are capable of assisting recombinant associated viruses (AAV) (rAAV) vector transfer for cartilage repair as such vectors are more effective than nonviral vehicles to deliver genetic material in target cells [10,11]
The reporter rAAV-lacZ gene vector was first formulated with the various CDs (CD-1 to CD-4) to examine the ability of these nanoparticles to associate with rAAV and release it over time using Cy labeling and fluorescent evaluation of the vectors in the systems and by measuring the rAAV concentrations in the culture medium via AAV titration ELISA

Summary

Introduction

Articular cartilage lesions represent serious clinical issues in orthopaedics as this specialized tissue does not fully heal on itself by lack of vascularization and of local chondroregenerative cells that may repopulate the defects [1,2]. Biomaterial-assisted rAAV gene transfer for cartilage research has been described including polymeric micelles [24,25,26,27], hydrogels [28,29,30,31,32] and solid scaffolds [33], yet other materials may constitute valuable systems for rAAV delivery in experimental cartilage therapy In this regard, carbon dots (CDs), a recently discovered class of carbon-dominated, biocompatible nanomaterials [34,35] used in drug delivery and theranostic approaches [35,36], may be good candidates to achieve this goal as they have been reported for their ability to intracellularly deliver nucleic acids and proteins in vitro [37] and in experimental models in vivo of cancer [38,39,40] and for regenerative medicine [41,42]. It remains to be seen whether CDs are capable of assisting rAAV vector transfer for cartilage repair as such vectors are more effective than nonviral vehicles to deliver genetic material in target cells [10,11]

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Methods

Results