**1. Introduction**

Osteoarthritis (OA) represents a prevalent, chronic, and deteriorating joint affliction that is the leading cause of impaired function and disability [1]. OA is characterized by multiple functional and structural cartilage tissue and cell shifts, such as the progressive and permanent degradation of the articular cartilage matrix (loss of type-II collagen and of proteoglycans), the restructuration of the subchondral bone, and the formation of osteophytes [2,3] due to defective homeostasis [4,5]. Of note, none of the current pharmacological options and surgical alternatives [1] for treating OA can reestablish the native cartilage quality in patients.

Current research associates the changes observed in OA disease with a complex cascade of biochemical factors, including proteolytic enzymes that promote the disruption of the cartilage macromolecules [6]. Pro-inflammatory cytokines such as interleukin 1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α), produced by mononuclear cells, activated synoviocytes or by the cartilage itself, upregulate metalloproteinases gene expression, impairing chondrocyte counteracting synthetic pathways necessary to reinstate the integrity of the degenerated extracellular matrix (ECM) [6].

In this context, previous studies have shown an abolishment of type-II collagen expression from primary human articular chondrocytes via suppression of the expression of the cartilage-associated sex-determining region Y-type high mobility box 9 (SOX9) transcription factor upon treatment with IL-1β [7]. Of note, overexpression of *sox9* via lentiviral vector has already been shown to preserve chondrocytes from IL-1β-induced apoptosis and degeneration [8]. However, while efficient, lentiviral vectors are not well adapted for translational approaches, as they involve a risk of insertional mutagenesis upon integration into the genome of host cells [9]. In contrast, recombinant adeno-associated viral (rAAV) vectors mainly remain episomal in the nucleus of their targets, showing potential integration events at very low frequency (0.1–1% vide infra) [10], while also allowing for highly effective gene transfer efficiencies even in nondividing cells like articular chondrocytes (more than 70%) [11]. rAAV vectors have thus emerged as the preferred gene carriers in several regenerative medicine applications including for cartilage repair [12–16].

A high and prolonged gene transmission efficiency in articular chondrocytes both in vitro and through their compact ECM in situ has been reported via rAAV vectors (up to 80% for at least 150 days) has been reported [11]. Furthermore, gene transfer of an rAAV TGF-β vector has been shown to promote the biological activities both in human articular chondrocytes cultures in vitro and in articular cartilage explants in situ [17,18]. In addition, overexpression of *sox9* via rAAV led to increased levels of type-II collagen and proteoglycans in both normal and OA-affected articular chondrocytes in vitro [19].

Still, administration of rAAV vectors in patients may be hampered by the prevalence of anti-AAV antibodies directed against viral capsid proteins in individuals as those prevailing in synovial fluid from patients affected with joint disorders [20]. We previously described the suitability of rAAV vectors (*lacZ*) encapsulation in poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO)-based polymeric micelles from linear (poloxamers; PF68) or X-shaped copolymers (poloxamines; T908), as a way to overcome such obstacles while affording protection to the vectors in experimental settings of neutralization and increasing their gene transfer efficacy [21,22]. Interestingly, overexpression of *sox9* using such systems resulted in the effective remodeling of human OA cartilage, leading to increases in cell proliferation activities and in proteoglycan deposition relative to free vector administration [23]. Yet, it remains to be seen whether such micellar systems can also be efficient for delivering rAAV vectors and overexpressing their transgenes in an inflammatory, detrimental environment like in OA (IL-1β, TNF-α) [4,5,24].

The aim of the present study was therefore to test the ability of PF68- and T908-based polymeric micelles to deliver the therapeutic rAAV-FLAG-h*sox9* candidate vector in human OA chondrocytes, the sole cell population present in the articular cartilage, in the presence of OA-associated pro-inflammatory cytokines (IL-1β, TNF-α) in a 2D environment as a preliminary proof of concept, as a means to effectively restore the chondrocyte phenotype in such cells in vitro.
