Niosomes-based gene delivery systems for effective transfection of human mesenchymal stem cells.

Gene transfer to mesenchymal stem cells (MSCs) has arisen as a powerful approach to increase the therapeutic potential of this effective cell population. Over recent years, niosomes have emerged as self-assembled carriers with promising performance for gene delivery. The aim of our work was to develop effective niosomes-based DNA delivery platforms for targeting MSCs. Niosomes based on 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA; 0, 7 or 15%) as cationic lipid, cholesterol as helper lipid, and polysorbate 60 as non-ionic surfactant, were prepared using a reverse phase evaporation technique. Niosomes dispersions (filtered or not) and their corresponding nioplexes with a lacZ plasmid were characterized in terms of size, charge, protection, and complexation abilities. DOTMA concentration had a large influence on the physicochemical properties of resulting nioplexes. Transfection efficiency and cytotoxic profiles were assessed in two immortalized cell lines of MSCs. Niosomes formulated with 15% DOTMA provided the highest values of ß-galactosidase activity, being similar to those achieved with Lipofectamine®, but showed less cytotoxicity. Filtration of niosomes dispersions before adding to the cells resulted in a loss of their biological activities. Storage of niosomes formulations (for 30 days at room temperature) caused minor modification of their physicochemical properties but also attenuated the transfection capability of the nioplexes. Differently, addition of the lysosomotropic agent sucrose into the culture medium during transfection or to the formulation itself improved the transfection performance of non-filtered niosomes. Indeed, steam heat-sterilized niosomes prepared in sucrose medium demonstrated transfection capability.

Hydrogel-Guided, rAAV-Mediated IGF-I Overexpression Enables Long-Term Cartilage Repair and Protection against Perifocal Osteoarthritis in a Large-Animal Full-Thickness Chondral Defect Model at One Year In Vivo.

The regeneration of focal articular cartilage defects is complicated by the reduced quality of the repair tissue and the potential development of perifocal osteoarthritis (OA). Biomaterial-guided gene therapy may enhance cartilage repair by controlling the release of therapeutic sequences in a spatiotemporal manner. Here, the benefits of delivering a recombinant adeno-associated virus (rAAV) vector coding for the human insulin-like growth factor I (IGF-I) via an alginate hydrogel (IGF-I/AlgPH155) to enhance repair of full-thickness chondral defects following microfracture surgery after one year in minipigs versus control (lacZ/AlgPH155) treatment are reported. Sustained IGF-I overexpression is significantly achieved in the repair tissue of defects treated with IGF-I/AlgPH155 versus those receiving lacZ/AlgPH155 for one year and in the cartilage surrounding the defects. Administration of IGF-I/AlgPH155 significantly improves parameters of cartilage repair at one year relative to lacZ/AlgPH155 (semiquantitative total histological score, cell densities, matrix deposition) without deleterious or immune reactions. Remarkably, delivery of IGF-I/AlgPH155 also significantly reduces perifocal OA and inflammation after one year versus lacZ/AlgPH155 treatment. Biomaterial-guided rAAV gene transfer represents a valuable clinical approach to promote cartilage repair and to protect against OA.

pNaSS-Grafted PCL Film-Guided rAAV TGF-ß Gene Therapy Activates the Chondrogenic Activities in Human Bone Marrow Aspirates.

Scaffold-guided viral gene therapy is a novel, powerful tool to enhance the processes of tissue repair in articular cartilage lesions by the delivery and overexpression of therapeutic genes in a noninvasive, controlled release manner based on a procedure that may protect the gene vehicles from undesirable host immune responses. In this study, we examined the potential of transferring a recombinant adeno-associated virus (rAAV) vector carrying a sequence for the highly chondroregenerative transforming growth factor beta (TGF-ß), using poly(ɛ-caprolactone) (PCL) films functionalized by the grafting of poly(sodium styrene sulfonate) (pNaSS) in chondrogenically competent bone marrow aspirates as future targets for therapy in cartilage lesions. Effective overexpression of TGF-ß in the aspirates by rAAV was achieved upon delivery using pNaSS-grafted and ungrafted PCL films for up to 21 days (the longest time point evaluated), with superior levels using the grafted films, compared with respective conditions without vector coating. The production of rAAV-mediated TGF-ß by pNaSS-grafted and ungrafted PCL films significantly triggered the biological activities and chondrogenic processes in the samples (proteoglycan and type-II collagen deposition and cell proliferation), while containing premature mineralization and hypertrophy relative to the other conditions, with overall superior effects supported by the pNaSS-grafted films. These observations demonstrate the potential of PCL film-assisted rAAV TGF-ß gene transfer as a convenient, off-the-shelf technique to enhance the reparative potential of the bone marrow in patients in future approaches for improved cartilage repair.

Therapeutic Delivery of rAAV sox9 via Polymeric Micelles Counteracts the Effects of Osteoarthritis-Associated Inflammatory Cytokines in Human Articular Chondrocytes.

Osteoarthritis (OA) is a prevalent joint disease linked to the irreversible degradation of key extracellular cartilage matrix (ECM) components (proteoglycans, type-II collagen) by proteolytic enzymes due to an impaired tissue homeostasis, with the critical involvement of OA-associated pro-inflammatory cytokines (interleukin 1 beta, i.e., IL-1ß, and tumor necrosis factor alpha, i.e., TNF-α). Gene therapy provides effective means to re-establish such degraded ECM compounds by rejuvenating the altered OA phenotype of the articular chondrocytes, the unique cell population ubiquitous in the articular cartilage. In particular, overexpression of the highly specialized SOX9 transcription factor via recombinant adeno-associated viral (rAAV) vectors has been reported for its ability to readjust the metabolic balance in OA, in particular via controlled rAAV delivery using polymeric micelles as carriers to prevent a possible vector neutralization by antibodies present in the joints of patients. As little is known on the challenging effects of such naturally occurring OA-associated pro-inflammatory cytokines on such rAAV/polymeric gene transfer, we explored the capacity of polyethylene oxide (PEO) and polypropylene oxide (PPO)-based polymeric micelles to deliver a candidate rAAV-FLAG-hsox9 construct in human OA chondrocytes in the presence of IL-1ß and TNF-α. We report that effective, micelle-guided rAAV sox9 overexpression enhanced the deposition of ECM components and the levels of cell survival, while advantageously reversing the deleterious effects afforded by the OA cytokines on these processes. These findings highlight the potentiality of polymeric micelles as effective rAAV controlled delivery systems to counterbalance the specific contribution of major OA-associated inflammatory cytokines, supporting the concept of using such systems for the treatment for chronic inflammatory diseases like OA.

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

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-ß), 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-ß) rAAV vectors in hMSCs via CD-2 led to the effective overexpression of each independent transgene, promoting enhanced cell proliferation (TGF-ß) 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.

Controlled Release of rAAV Vectors from APMA-Functionalized Contact Lenses for Corneal Gene Therapy.

As an alternative to eye drops and ocular injections for gene therapy, the aim of this work was to design for the first time hydrogel contact lenses that can act as platforms for the controlled delivery of viral vectors (recombinant adeno-associated virus, rAAV) to the eye in an effective way with improved patient compliance. Hydrogels of hydroxyethyl methacrylate (HEMA) with aminopropyl methacrylamide (APMA) (H1: 40, and H2: 80 mM) or without (Hc: 0 mM) were synthesized, sterilized by steam heat (121 °C, 20 min), and then tested for gene therapy using rAAV vectors to deliver the genes to the cornea. The hydrogels showed adequate light transparency, oxygen permeability, and swelling for use as contact lenses. Loading of viral vectors (rAAV-lacZ, rAAV-RFP, or rAAV-hIGF-I) was carried out at 4 °C to maintain viral vector titer. Release in culture medium was monitored by fluorescence with Cy3-rAAV-lacZ and AAV Titration ELISA. Transduction efficacy was tested through reporter genes lacZ and RFP in human bone marrow derived mesenchymal stem cells (hMSCs). lacZ was detected with X-Gal staining and quantified with Beta-Glo®, and RFP was monitored by fluorescence. The ability of rAAV-hIGF-I-loaded hydrogels to trigger cell proliferation in hMSCs was evaluated by immunohistochemistry. Finally, the ability of rAAV-lacZ-loaded hydrogels to transduce bovine cornea was confirmed through detection with X-Gal staining of ß-galactosidase expressed within the tissue.

Enhanced Chondrogenic Differentiation Activities in Human Bone Marrow Aspirates via sox9 Overexpression Mediated by pNaSS-Grafted PCL Film-Guided rAAV Gene Transfer.

BACKGROUND: The delivery of therapeutic genes in sites of articular cartilage lesions using non-invasive, scaffold-guided gene therapy procedures is a promising approach to stimulate cartilage repair while protecting the cargos from detrimental immune responses, particularly when targeting chondroreparative bone marrow-derived mesenchymal stromal cells in a natural microenvironment like marrow aspirates. METHODS: Here, we evaluated the benefits of providing a sequence for the cartilage-specific sex-determining region Y-type high-mobility group box 9 (SOX9) transcription factor to human marrow aspirates via recombinant adeno-associated virus (rAAV) vectors delivered by poly(ε-caprolactone) (PCL) films functionalized via grafting with poly(sodium styrene sulfonate) (pNaSS) to enhance the marrow chondrogenic potential over time. RESULTS: Effective sox9 overexpression was observed in aspirates treated with pNaSS-grafted or ungrafted PCL films coated with the candidate rAAV-FLAG-hsox9 (FLAG-tagged rAAV vector carrying a human sox9 gene sequence) vector for at least 21 days relative to other conditions (pNaSS-grafted and ungrafted PCL films without vector coating). Overexpression of sox9 via rAAV sox9/pNaSS-grafted or ungrafted PCL films led to increased biological and chondrogenic differentiation activities (matrix deposition) in the aspirates while containing premature osteogenesis and hypertrophy without impacting cell proliferation, with more potent effects noted when using pNaSS-grafted films. CONCLUSIONS: These findings show the benefits of targeting patients' bone marrow via PCL film-guided therapeutic rAAV (sox9) delivery as an off-the-shelf system for future strategies to enhance cartilage repair in translational applications.

Thermosensitive Hydrogel Based on PEO-PPO-PEO Poloxamers for a Controlled In Situ Release of Recombinant Adeno-Associated Viral Vectors for Effective Gene Therapy of Cartilage Defects.

Advanced biomaterial-guided delivery of gene vectors is an emerging and highly attractive therapeutic solution for targeted articular cartilage repair, allowing for a controlled and minimally invasive delivery of gene vectors in a spatiotemporally precise manner, reducing intra-articular vector spread and possible loss of the therapeutic gene product. As far as it is known, the very first successful in vivo application of such a biomaterial-guided delivery of a potent gene vector in an orthotopic large animal model of cartilage damage is reported here. In detail, an injectable and thermosensitive hydrogel based on poly(ethylene oxide) (PEO)-poly(propylene oxide) (PPO)-PEO poloxamers, capable of controlled release of a therapeutic recombinant adeno-associated virus (rAAV) vector overexpressing the chondrogenic sox9 transcription factor in full-thickness chondral defects, is applied in a clinically relevant minipig model in vivo. These comprehensive analyses of the entire osteochondral unit with multiple standardized evaluation methods indicate that rAAV-FLAG-hsox9/PEO-PPO-PEO hydrogel-augmented microfracture significantly improves cartilage repair with a collagen fiber orientation more similar to the normal cartilage and protects the subchondral bone plate from early bone loss.

Effects of rAAV-Mediated sox9 Overexpression on the Biological Activities of Human Osteoarthritic Articular Chondrocytes in Their Intrinsic Three-Dimensional Environment.

Gene therapy for osteoarthritis offers powerful, long-lasting tools that are well adapted to treat such a slow, progressive disorder, especially those therapies based on the clinically adapted recombinant adeno-associated viral (rAAV) vectors. Here, we examined the ability of an rAAV construct carrying a therapeutic sequence for the cartilage-specific SOX9 transcription factor to modulate the phenotype of human osteoarthritic articular chondrocytes compared with normal chondrocytes in a three-dimensional environment where the cells are embedded in their extracellular matrix. Successful sox9 overexpression via rAAV was noted for at least 21 days, leading to the significant production of major matrix components (proteoglycans, type-II collagen) without affecting the proliferation of the cells, while the cells contained premature hypertrophic processes relative to control conditions (reporter rAAV-lacZ application, absence of vector treatment). These findings show the value of using rAAV to adjust the osteoarthritic phenotype when the chondrocytes are confined in their inherently altered environment and the possibility of impacting key cellular processes via gene therapy to remodel human osteoarthritic cartilage lesions.

Remodeling of Human Osteochondral Defects via rAAV-Mediated Co-Overexpression of TGF-ß and IGF-I from Implanted Human Bone Marrow-Derived Mesenchymal Stromal Cells.

The application of chondrogenic gene sequences to human bone marrow-derived mesenchymal stromal cells (hMSCs) is an attractive strategy to activate the reparative activities of these cells as a means to enhance the processes of cartilage repair using indirect cell transplantation procedures that may improve the repopulation of cartilage lesions. In the present study, we examined the feasibility of co-delivering the highly competent transforming growth factor beta (TGF-ß) with the insulin-like growth factor I (IGF-I) in hMSCs via recombinant adeno-associated virus (rAAV) vector-mediated gene transfer prior to implantation in a human model of osteochondral defect (OCD) ex vivo that provides a microenvironment similar to that of focal cartilage lesions. The successful co-overexpression of rAAV TGF-ß/IGF-I in implanted hMSCs promoted the durable remodeling of tissue injury in human OCDs over a prolonged period of time (21 days) relative to individual gene transfer and the control (reporter lacZ gene) treatment, with enhanced levels of cell proliferation and matrix deposition (proteoglycans, type-II collagen) both in the lesions and at a distance, while hypertrophic, osteogenic, and catabolic processes could be advantageously delayed. These findings demonstrate the value of indirect, progenitor cell-based combined rAAV gene therapy to treat human focal cartilage defects in a natural environment as a basis for future clinical applications.

Current Trends in Viral Gene Therapy for Human Orthopaedic Regenerative Medicine.

Background: Viral vector-based therapeutic gene therapy is a potent strategy to enhance the intrinsic reparative abilities of human orthopaedic tissues. However, clinical application of viral gene transfer remains hindered by detrimental responses in the host against such vectors (immunogenic responses, vector dissemination to nontarget locations). Combining viral gene therapy techniques with tissue engineering procedures may offer strong tools to improve the current systems for applications in vivo. Methods: The goal of this work is to provide an overview of the most recent systems exploiting biomaterial technologies and therapeutic viral gene transfer in human orthopaedic regenerative medicine. Results: Integration of tissue engineering platforms with viral gene vectors is an active area of research in orthopaedics as a means to overcome the obstacles precluding effective viral gene therapy. Conclusions: In light of promising preclinical data that may rapidly expand in a close future, biomaterial-guided viral gene therapy has a strong potential for translation in the field of human orthopaedic regenerative medicine.

Therapeutic Effects of rAAV-Mediated Concomittant Gene Transfer and Overexpression of TGF-ß and IGF-I on the Chondrogenesis of Human Bone-Marrow-Derived Mesenchymal Stem Cells.

Application of chondroreparative gene vectors in cartilage defects is a powerful approach to directly stimulate the regenerative activities of bone-marrow-derived mesenchymal stem cells (MSCs) that repopulate such lesions. Here, we investigated the ability of combined recombinant adeno-associated virus (rAAV) vector-mediated delivery of the potent transforming growth factor beta (TGF-ß) and insulin-like growth factor I (IGF-I) to enhance the processes of chondrogenic differentiation in human MSCs (hMSCs) relative to individual candidate treatments and to reporter (lacZ) gene condition. The rAAV-hTGF-ß and rAAV-hIGF-I vectors were simultaneously provided to hMSC aggregate cultures (TGF-ß/IGF-I condition) in chondrogenic medium over time (21 days) versus TGF-ß/lacZ, IGF-I/lacZ, and lacZ treatments at equivalent vector doses. The cultures were then processed to monitor transgene (co)-overexpression, the levels of biological activities in the cells (cell proliferation, matrix synthesis), and the development of a chondrogenic versus osteogenic/hypertrophic phenotype. Effective, durable co-overexpression of TGF-ß with IGF-I via rAAV enhanced the proliferative, anabolic, and chondrogenic activities in hMSCs versus lacZ treatment and reached levels that were higher than those achieved upon single candidate gene transfer, while osteogenic/hypertrophic differentiation was delayed over the period of time evaluated. These findings demonstrate the potential of manipulating multiple therapeutic rAAV vectors as a tool to directly target bone-marrow-derived MSCs in sites of focal cartilage defects and to locally enhance the endogenous processes of cartilage repair.

Supramolecular Cyclodextrin-Based Hydrogels for Controlled Gene Delivery.

Controlled delivery of gene transfer vectors is a powerful strategy to enhance the temporal and spatial presentation of therapeutic agents in a defined target. Hydrogels are adapted biomaterials for gene delivery capable of acting as a localized depot of genes while maintaining the long term local availability of DNA vectors at a specific location. Supramolecular hydrogels based on cyclodextrins (CDs) have attracted considerable attention as potential biomaterials in a broad range of drug delivery applications. Their unique characteristics of thixotropicity and low cytotoxicity due to their production under mild conditions make them potential candidates to form injectable delivery systems. This work aims to provide an overview of the use of CD-based polypseudorotaxane hydrogels as controlled gene delivery systems for different applications in regenerative medicine.

Improved Chondrogenic Differentiation of rAAV SOX9-Modified Human MSCs Seeded in Fibrin-Polyurethane Scaffolds in a Hydrodynamic Environment.

The repair of focal articular cartilage defects remains a problem. Combining gene therapy with tissue engineering approaches using bone marrow-derived mesenchymal stem cells (MSCs) may allow the development of improved options for cartilage repair. Here, we examined whether a three-dimensional fibrin-polyurethane scaffold provides a favorable environment for the effective chondrogenic differentiation of human MSCs (hMSCs) overexpressing the cartilage-specific SOX9 transcription factor via recombinant adeno-associated virus (rAAV) -mediated gene transfer cultured in a hydrodynamic environment in vitro. Sustained SOX9 expression was noted in the constructs for at least 21 days, the longest time point evaluated. Such spatially defined SOX9 overexpression enhanced proliferative, metabolic, and chondrogenic activities compared with control (reporter lacZ gene transfer) treatment. Of further note, administration of the SOX9 vector was also capable of delaying premature hypertrophic and osteogenic differentiation in the constructs. This enhancement of chondrogenesis by spatially defined overexpression of human SOX9 demonstrate the potential benefits of using rAAV-modified hMSCs seeded in fibrin-polyurethane scaffolds as a promising approach for implantation in focal cartilage lesions to improve cartilage repair.

Chondrogenic Differentiation Processes in Human Bone-Marrow Aspirates Seeded in Three-Dimensional-Woven Poly(ɛ-Caprolactone) Scaffolds Enhanced by Recombinant Adeno-Associated Virus-Mediated SOX9 Gene Transfer.

Combining gene therapy approaches with tissue engineering procedures is an active area of translational research for the effective treatment of articular cartilage lesions, especially to target chondrogenic progenitor cells such as those derived from the bone marrow. This study evaluated the effect of genetically modifying concentrated human mesenchymal stem cells from bone marrow to induce chondrogenesis by recombinant adeno-associated virus (rAAV) vector gene transfer of the sex-determining region Y-type high-mobility group box 9 (SOX9) factor upon seeding in three-dimensional-woven poly(ɛ-caprolactone; PCL) scaffolds that provide mechanical properties mimicking those of native articular cartilage. Prolonged, effective SOX9 expression was reported in the constructs for at least 21 days, the longest time point evaluated, leading to enhanced metabolic and chondrogenic activities relative to the control conditions (reporter lacZ gene transfer or absence of vector treatment) but without affecting the proliferative activities in the samples. The application of the rAAV SOX9 vector also prevented undesirable hypertrophic and terminal differentiation in the seeded concentrates. As bone marrow is readily accessible during surgery, such findings reveal the therapeutic potential of providing rAAV-modified marrow concentrates within three-dimensional-woven PCL scaffolds for repair of focal cartilage lesions.

Effective Remodelling of Human Osteoarthritic Cartilage by sox9 Gene Transfer and Overexpression upon Delivery of rAAV Vectors in Polymeric Micelles.

Recombinant adeno-associated virus (rAAV) vectors are well suited carriers to provide durable treatments for human osteoarthritis (OA). Controlled release of rAAV from polymeric micelles was already shown to increase both the stability and bioactivity of the vectors while overcoming barriers, precluding effective gene transfer. In the present study, we examined the convenience of delivering rAAV vectors via poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) polymeric (PEO-PPO-PEO) micelles to transfer and overexpress the transcription factor SOX9 in monolayers of human OA chondrocytes and in experimentally created human osteochondral defects. Human osteoarthritic (OA) chondrocytes and human osteochondral defect models were produced using human OA cartilage obtained from patients subjected to total knee arthroplasty. Samples were genetically modified by adding a rAAV-FLAG-h sox9 vector in its free form or via polymeric micelles for 10 days relative to control conditions (unmodified cells). The effects of sox9 overexpression in human OA cartilage samples were monitored by biochemical, histological, and immunohistochemical analyses. Delivery of rAAV-FLAG-h sox9 via polymeric micelles enhanced the levels of sox9 expression compared with free vector administration, resulting in increased proteoglycan deposition and in a stimulated cell proliferation index in OA chondrocytes. Moreover, higher production of type II collagen and decreased hypertrophic events were noted in osteochondral defect cultures when compared with control conditions. Controlled therapeutic rAAV sox9 gene delivery using PEO-PPO-PEO micelles is a promising, efficient tool to promote the remodelling of human OA cartilage.

rAAV-mediated overexpression of TGF-ß via vector delivery in polymeric micelles stimulates the biological and reparative activities of human articular chondrocytes in vitro and in a human osteochondral defect model.

Recombinant adeno-associated virus (rAAV) vectors are clinically adapted vectors to durably treat human osteoarthritis (OA). Controlled delivery of rAAV vectors via polymeric micelles was reported to enhance the temporal and spatial presentation of the vectors into their targets. Here, we tested the feasibility of delivering rAAV vectors via poly (ethylene oxide) (PEO) and poly (propylene oxide) (PPO) (poloxamer and poloxamine) polymeric micelles as a means to overexpress the therapeutic factor transforming growth factor-beta (TGF-ß) in human OA chondrocytes and in experimental human osteochondral defects. Application of rAAV-human transforming growth factor-beta using such micelles increased the levels of TGF-ß transgene expression compared with free vector treatment. Overexpression of TGF-ß with these systems resulted in higher proteoglycan deposition and increased cell numbers in OA chondrocytes. In osteochondral defect cultures, a higher deposition of type-II collagen and reduced hypertrophic events were noted. Delivery of therapeutic rAAV vectors via PEO-PPO-PEO micelles may provide potential tools to remodel human OA cartilage.

Impact of mechanical stimulation on the chondrogenic processes in human bone marrow aspirates modified to overexpress sox9 via rAAV vectors.

BACKGROUND: Evaluation of gene-based approaches to target human bone marrow aspirates in conditions of mechanical stimulation that aim at reproducing the natural joint environment may allow to develop improved treatments for articular cartilage injuries. In the present study, we investigated the potential of rAAV-mediated sox9 gene transfer to enhance the chondrogenic differentiation processes in human bone marrow aspirates under established hydrodynamic conditions compared with the more commonly employed static culture conditions. METHODS: Fresh human bone marrow aspirates were transduced with rAAV-FLAG-hsox9 (40 µl) and maintained for up to 28 days in chondrogenic medium under mechanically-induced conditions in dynamic flow rotating bioreactors that permit tissue growth and matrix deposition relative to static culture conditions. The samples were then processed to examine the potential effects of sox9 overexpression on the cellular activities (matrix synthesis, proliferation) and on the chondrogenic differentiation potency compared with control treatments (absence of rAAV vector; reporter rAAV-lacZ, rAAV-RFP, and rAAV-luc gene transfer). RESULTS: Prolonged, significant sox9 overexpression via rAAV was achieved in the aspirates for at least 28 days when applying the rAAV-FLAG-hsox9 construct, leading to higher, prolonged levels of matrix biosynthesis and to enhanced chondrogenic activities relative to control treatments especially when maintaining the samples under mechanical stimulation. Administration of sox9 however did not impact the indices of proliferation in the aspirates. Remarkably, sox9 gene transfer also durably delayed hypertrophic and osteogenic differentiation in the samples regardless of the conditions of culture applied versus control treatments. CONCLUSIONS: The current observations show the value of genetically modifying human bone marrow aspirates upon mechanical stimulation by rAAV sox9 as a promising strategy for future treatments to improve cartilage repair by implantation in lesions where the tissue is submitted to natural mechanical forces.

Peripheral blood aspirates overexpressing IGF-I via rAAV gene transfer undergo enhanced chondrogenic differentiation processes.

Implantation of peripheral blood aspirates induced towards chondrogenic differentiation upon genetic modification in sites of articular cartilage injury may represent a powerful strategy to enhance cartilage repair. Such a single-step approach may be less invasive than procedures based on the use of isolated or concentrated MSCs, simplifying translational protocols in patients. In this study, we provide evidence showing the feasibility of overexpressing the mitogenic and pro-anabolic insulin-like growth factor I (IGF-I) in human peripheral blood aspirates via rAAV-mediated gene transfer, leading to enhanced proliferative and chondrogenic differentiation (proteoglycans, type-II collagen, SOX9) activities in the samples relative to control (reporter rAAV-lacZ) treatment over extended periods of time (at least 21 days, the longest time-point evaluated). Interestingly, IGF-I gene transfer also triggered hypertrophic, osteo- and adipogenic differentiation processes in the aspirates, suggesting that careful regulation of IGF-I expression may be necessary to contain these events in vivo. Still, the current results demonstrate the potential of targeting human peripheral blood aspirates via therapeutic rAAV transduction as a novel, convenient tool to treat articular cartilage injuries.

Smart and Controllable rAAV Gene Delivery Carriers in Progenitor Cells for Human Musculoskeletal Regenerative Medicine with a Focus on the Articular Cartilage.

Cell therapy using mesenchymal stem cells (MSCs) is a powerful tool for the treatment of various diseases and injuries. Still, important limitations including the large amounts of cells required for application in vivo and the age-related decline in lifespan, proliferation, and potency may hinder the use of MSCs in patients. In this regard, gene therapy may offer strong approaches to optimize the use of MSCs for regenerative medicine. Diverse nonviral and viral gene vehicles have been manipulated to genetically modify MSCs, among which the highly effective and relatively safe recombinant adeno-associated viral (rAAV) vectors that emerged as the preferred gene delivery system to treat human disorders. Yet, clinical adaptation of such gene vehicles may be limited by several hurdles, including the possibility of dissemination to nontarget sites and the presence of immune and toxic responses in the host organism that may impair their therapeutic actions. The use of smart biomaterials acting as interfaces to enhance the temporal and spatial presentation of therapeutic agents in the target place and/or acting as scaffolding for MSC growth is an innovative, valuable approach to overcome these shortcomings that else restrain the efficacy of such potent cell populations. Here, we provide an overview on the most recent tissue engineering approaches based on the use of biomaterials acting as vehicles for rAAV vectors to target MSCs directly in the recipient (in vivo strategy) or as supportive matrices for rAAV-modified MSCs for indirect cell reimplantation (ex vivo strategy) as means to activate the reparative processes in tissues of the musculoskeletal system.