Advances in Human Mitochondria-Based Therapies

Mitochondria are the key biological generators of eukaryotic cells, controlling the energy supply while providing many important biosynthetic intermediates. Mitochondria act as a dynamic, functionally and structurally interconnected network hub closely integrated with other cellular compartments via biomembrane systems, transmitting biological information by shuttling between cells and tissues. Defects and dysregulation of mitochondrial functions are critically involved in pathological mechanisms contributing to aging, cancer, inflammation, neurodegenerative diseases, and other severe human diseases. Mediating and rejuvenating the mitochondria may therefore be of significant benefit to prevent, reverse, and even treat such pathological conditions in patients. The goal of this review is to present the most advanced strategies using mitochondria to manage such disorders and to further explore innovative approaches in the field of human mitochondriabased therapies

Is Extracellular Vesicle-Based Therapy the Next Answer for Cartilage Regeneration?

“Extracellular vesicles” (EVs) is a term gathering biological particles released from cells that act as messengers for cell-to-cell communication. Like cells, EVs have a membrane with a lipid bilayer, but unlike these latter, they have no nucleus and consequently cannot replicate. Several EV subtypes (e.g., exosomes, microvesicles) are described in the literature. However, the remaining lack of consensus on their specific markers prevents sometimes the full knowledge of their biogenesis pathway, causing the authors to focus on their biological effects and not their origins. EV signals depend on their cargo, which can be naturally sourced or altered (e.g., cell engineering). The ability for regeneration of adult articular cartilage is limited because this avascular tissue is partly made of chondrocytes with a poor proliferation rate and migration capacity. Mesenchymal stem cells (MSCs) had been extensively used in numerous in vitro and preclinical animal models for cartilage regeneration, and it has been demonstrated that their therapeutic effects are due to paracrine mechanisms involving EVs. Hence, using MSC-derived EVs as cell-free therapy tools has become a new therapeutic approach to improve regenerative medicine. EV-based therapy seems to show similar cartilage regenerative potential compared with stem cell transplantation without the associated hindrances (e.g., chromosomal aberrations, immunogenicity). The aim of this short review is to take stock of occurring EV-based treatments for cartilage regeneration according to their healing effects. The article focuses on cartilage regeneration through various sources used to isolate EVs (mature or stem cells among others) and beneficial effects depending on cargos produced from natural or tuned EVs

EF hand-mediated Ca2+- and cGMP-signaling in photoreceptor synaptic terminals

Photoreceptors, the light-sensitive receptor neurons of the retina, receive and transmit a plethora of visual informations from the surrounding world. Photoreceptors capture light and convert this energy into electrical signals that are conveyed to the inner retina. For synaptic communication with the inner retina, photoreceptors make large active zones that are marked by synaptic ribbons. These unique synapses support continuous vesicle exocytosis that is modulated by light-induced, graded changes of membrane potential. Synaptic transmission can be adjusted in an activity-dependent manner, and at the synaptic ribbons, Ca2+- and cGMP-dependent processes appear to play a central role. EF-hand-containing proteins mediate many of these Ca2+- and cGMP-dependent functions. Since continuous signaling of photoreceptors appears to be prone to malfunction, disturbances of Ca2+- and cGMP-mediated signaling in photoreceptors can lead to visual defects, retinal degeneration (rd), and even blindness. This review summarizes aspects of signal transmission at the photoreceptor presynaptic terminals that involve EF-hand-containing Ca2+-binding proteins

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

Cytotoxic effects of different mouthwash solutions on primary human articular chondrocytes and normal human articular cartilage : an in vitro study

Objectives To compare the cytotoxicity of octenidine dihydrochloride and chlorhexidine gluconate at diferent concentrations on primary human articular chondrocytes and cartilage. Materials and methods Primary cultures of human normal adult articular chondrocytes were exposed to octenidine dihydrochloride (0.001562%, 0.003125%, 0.00625%, 0.0125%, 0.025%, 0.05%, and 0.1%), chlorhexidine gluconate (0.003125%, 0.00625%, 0.0125%, 0.025%, 0.05%, 0.1%, and 0.2%), and control (Dulbecco’s modifed Eagle medium or phosphate-bufered saline) for 30 s. Normal human articular cartilage explants were exposed to octenidine dihydrochloride (0.1% versus control) and chlorhexidine gluconate (0.1% versus control) for 30 s. The viability of human articular chondrocytes was measured by Trypan blue staining, Cell Proliferation Reagent WST-1, and Live/Dead staining. The proliferation of human chondrocytes was measured using the Cell Proliferation Reagent WST-1. The viability of human articular cartilage explants was measured by using Live/Dead staining. Results Octenidine dihydrochloride and chlorhexidine gluconate exposure decreased cell viability and proliferation in a dose-dependent manner in primary human articular chondrocytes. Octenidine dihydrochloride and chlorhexidine gluconate exposure decreased cell viability in human articular cartilage explant cultures. Conclusion The degree of toxicity varied between octenidine dihydrochloride and chlorhexidine gluconate, with chlorhexidine gluconate being less toxic than octenidine dihydrochloride at the same concentration. Additionally, both octenidine dihydrochloride and chlorhexidine gluconate evaluation had cytotoxic efects on human articular cartilage. Therefore, dosing for the antimicrobial mouthwash ingredients administration would ideally be determined to remain below IC50. Clinical relevance These data support the in vitro safety of antimicrobial mouthwashes on primary adult human articular chondrocytes

Effects of rAAV-Mediated Overexpression of sox9 and TGF-ß via Alginate Hydrogel-Guided Vector Delivery on the Chondroreparative Activities of Human Bone Marrow-Derived Mesenchymal Stromal Cells

Recombinant adeno-associated virus (rAAV) vectors have a strong potential to promote the healing of traumatic cartilage defects and osteoarthritic lesions upon delivery and overexpression of therapeutic genes from suitable biomaterials that support a controlled release of the candidate constructs. Te goal of the present work is to examine whether the administration of chondrogenic rAAV sox9 and rAAV TGF-ß gene vehicles via alginate hydrogel-guided vector delivery stimulates the biological and chondroreparative activities of human bone marrow-derived mesenchymal stromal cells (hMSCs) as a source of improved reparative cells for future implantation in sites of cartilage damage. Te delivery of rAAV using an alginate (AlgPH155) hydrogel system is successfully achieved in hMSCs over time (21 days), leading to the efective overexpression of sox9 and TGF-ß that signifcantly increases the proliferation and chondrogenic diferentiation activities of the cells relative to control (marker lacZ) gene transfer while advantageously preventing premature hypertrophy, osteogenesis, and mineralization. Tis study reveals the potential of alginate hydrogel-based systems to control the delivery of rAAV (sox9 and TGF-ß) gene vectors to adeptly trigger the chondroreparative activities of hMSCs for future applications that aim at improving cartilage repair

Advanced Gene Therapy Strategies for the Repair of ACL Injuries

The anterior cruciate ligament (ACL), the principal ligament for stabilization of the knee, is highly predisposed to injury in the human population. As a result of its poor intrinsic healing capacities, surgical intervention is generally necessary to repair ACL lesions, yet the outcomes are never fully satisfactory in terms of long-lasting, complete, and safe repair. Gene therapy, based on the transfer of therapeutic genetic sequences via a gene vector, is a potent tool to durably and adeptly enhance the processes of ACL repair and has been reported for its workability in various experimental models relevant to ACL injuries in vitro, in situ, and in vivo. As critical hurdles to the effective and safe translation of gene therapy for clinical applications still remain, including physiological barriers and host immune responses, biomaterial-guided gene therapy inspired by drug delivery systems has been further developed to protect and improve the classical procedures of gene transfer in the future treatment of ACL injuries in patients, as critically presented here

rAAV TGF-β and FGF-2 Overexpression via pNaSS-Grafted PCL Films Stimulates the Reparative Activities of Human ACL Fibroblasts

Lesions in the human anterior cruciate ligament (ACL) are frequent, unsolved clinical issues due to the limited self-healing ability of the ACL and lack of treatments supporting full, durable ACL repair. Gene therapy guided through the use of biomaterials may steadily activate the processes of repair in sites of ACL injury. The goal of the present study was to test the hypothesis that functionalized poly(sodium styrene sulfonate)-grafted poly(ε-caprolactone) (pNaSS-grafted PCL) films can effectively deliver recombinant adeno-associated virus (rAAV) vectors as a means of overexpressing two reparative factors (transforming growth factor beta-TGF-β and basic fibroblast growth factor-FGF-2) in primary human ACL fibroblasts. Effective, durable rAAV reporter red fluorescent protein and candidate TGF-β and FGF-2 gene overexpression was achieved in the cells for at least 21 days, especially when pNaSS-grafted PCL films were used versus control conditions, such as ungrafted films and systems lacking vectors or films (between 1.8- and 5.2-fold differences), showing interactive regulation of growth factor production. The expression of TGF-β and FGF-2 from rAAV via PCL films safely enhanced extracellular matrix depositions of type-I/-III collagen, proteoglycans/decorin, and tenascin-C (between 1.4- and 4.5-fold differences) in the cells over time with increased levels of expression of the specific transcription factors Mohawk and scleraxis (between 1.7- and 3.7-fold differences) and without the activation of the inflammatory mediators IL-1β and TNF-α, most particularly with pNaSS-grafted PCL films relative to the controls. This work shows the value of combining rAAV gene therapy with functionalized PCL films to enhance ACL 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 β-galactosidase expressed within the tissue

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