Janina Frisch

Benefits of recombinant adeno-associated virus (rAAV)-mediated insulinlike growth factor I (IGF-I) overexpression for the long-term reconstruction of human osteoarthritic cartilage by modulation of the IGF-I axis

Administration of therapeutic genes to human osteoarthritic (OA) cartilage is a potential approach to generate effective, durable treatments against this slow, progressive disorder. Here, we tested the ability of recombinant adeno-associated virus (rAAV)-mediated overexpression of human insulinlike growth factor (hIGF)-I to reproduce an original surface in human OA cartilage in light of the pleiotropic activities of the factor. We examined the proliferative, survival and anabolic effects of the rAAV-hIGF-I treatment in primary human normal and OA chondrocytes in vitro and in explant cultures in situ compared with control (reporter) vector delivery. Efficient, prolonged IGF-I secretion via rAAV stimulated the biological activities of OA chondrocytes in all the systems evaluated over extended periods of time, especially in situ, where it allowed for the long-term reconstruction of OA cartilage (at least for 90 d). Remarkably, production of high, stable amounts of IGF-I in OA cartilage using rAAV advantageously modulated the expression of central effectors of the IGF-I axis by downregulating IGF-I inhibitors (IGF binding protein (IGFBP)-3 and IGFBP4) while upregulating key potentiators (IGFBP5, the IGF-I receptor and downstream mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (MAPK/ERK-1/2) and phosphatidylinisitol-3/Akt (PI3K/Akt) signal transduction pathways), probably explaining the enhanced responsiveness of OA cartilage to IGF-I treatment. These findings show the benefits of directly providing an IGF-I sequence to articular cartilage via rAAV for the future treatment of human osteoarthritis.

Determination of the chondrogenic differentiation processes in human bone marrow-derived mesenchymal stem cells genetically modified to overexpress transforming growth factor-β via recombinant adeno-associated viral vectors.

Genetic modification of bone marrow-derived mesenchymal stem cells (MSCs) for use in transplantation settings may be a valuable strategy to enhance the repair processes in articular cartilage defects. Here, we evaluated the potential of overexpressing the transforming growth factor (TGF)-β via recombinant adeno-associated viral (rAAV) vector-mediated gene transfer to promote the chondrogenic differentiation of human MSCs (hMSCs). A human TGF-β sequence was delivered to undifferentiated and chondrogenically induced primary hMSCs, using rAAV vectors to test the efficacy and duration of transgene expression and its effects on the chondrogenic, osteogenic, and adipogenic differentiation patterns of the cells compared with control (lacZ) treatment after 21 days in vitro. Significant, durable TGF-β expression was noted both in undifferentiated and chondrogenically induced hMSCs transduced with the candidate rAAV-hTGF-β vector for up to 21 days compared with rAAV-lacZ treatment, allowing for increased proliferative, metabolic, and chondrogenic activities via stimulation of the critical SOX9 (SRY [sex-determining region Y]-related HMG [high-mobility group] box 9) chondrogenic pathway. Overexpression of TGF-β under the conditions applied here also activated the hypertrophic and osteogenic differentiation processes in the treated cells. Such effects were noted in association with enhanced levels of β-catenin and Indian hedgehog and decreased parathyroid hormone-related protein expression. The current findings show that rAAV vectors provide advantageous vehicles for gene- and stem cell-based approaches to treat articular cartilage defects, requiring tight regulation of TGF-β expression to avoid hypertrophy as candidate treatment for future applications in clinically relevant animal models in vivo.

Effective and durable genetic modification of human mesenchymal stem cells via controlled release of rAAV vectors from self-assembling peptide hydrogels with a maintained differentiation potency.

Controlling the release of recombinant adeno-associated virus (rAAV) vectors from biocompatible materials is a novel, attractive approach to increase the residence time and effectiveness of a gene carrier at a defined target site. Self-assembling peptides have an ability to form stable hydrogels and encapsulate cells upon exposure to physiological pH and ionic strength. Here, we examined the capacity of the peptide hydrogel RAD16-I in a pure (RAD) form or combined with hyaluronic acid (RAD-HA) to release rAAV vectors as a means to genetically modify primary human bone marrow-derived mesenchymal stem cells (hMSCs), a potent source of cells for regenerative medicine. Specifically, we demonstrate the ability of the systems to efficiently encapsulate and release rAAV vectors in a sustained, controlled manner for the effective transduction of hMSCs (up to 80%) without deleterious effects on cell viability (up to 100%) or on their potential for chondrogenic differentiation over time (up to 21days). The present study demonstrates that RAD16-I is an advantageous material with tunable properties to control the release of rAAV vectors as a promising tool to develop new, improved therapeutic approaches for tissue engineering in vivo.

Current Progress in Stem Cell-Based Gene Therapy for Articular Cartilage Repair

Administration of mesenchymal stem cells (MSCs) that have a reliable potential for chondrogenic differentiation is a promising approach currently employed to treat articular cartilage lesions (focal defects and osteoarthritis) in patients as a mean to enhance the poor intrinsic capabilities of this specialized tissue for self-repair. However, there is still a critical need for improved designs, as reproduction of a native structural and functional unit in sites of cartilage damage is not occurring upon implantation of such cells. With the availability of optimized gene transfer systems, gene therapy offers powerful tools to stimulate the chondrogenic process in MSCs via the effective, safe, and durable delivery of candidate sequences with chondroprotective and/or chondroregenerative properties, both in vitro and in experimental models of cartilage lesions in vivo. In the present article, we provide an overview of the current advances in gene- and stem cell-based treatments employed to promote cartilage repair in focal defects and for osteoarthritis, and discuss the challenges that remain to be addressed for a safe translation of such procedures into the clinics.

PEO–PPO–PEO micelles as effective rAAV-mediated gene delivery systems to target human mesenchymal stem cells without altering their differentiation potency

UNLABELLED Recombinant adeno-associated viral (rAAV) vectors are clinically adapted gene transfer vectors for direct human cartilage regenerative medicine. Their appropriate use in patients is still limited by a relatively low efficacy of vector penetration inside the cells, by the pre-existing humoral immune responses against the viral capsid proteins in a large part of the human population, and by possible inhibition of viral uptake by clinical compounds such as heparin. The delivery of rAAV vectors to their targets using optimized vehicles is therefore under active investigation. Here, we evaluated the possibility of providing rAAV to human bone marrow-derived mesenchymal stem cells (hMSCs), a potent source of cartilage regenerative cells, via self-assembled poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) triblock copolymers as linear poloxamers or X-shaped poloxamines. Encapsulation in poloxamer PF68 and poloxamine T908 polymeric micelles allowed for an effective, durable, and safe modification of hMSCs via rAAV to levels similar to or even higher than those noted upon direct vector application. The copolymers were capable of restoring the transduction of hMSCs with rAAV in conditions of gene transfer inhibition, i.e. in the presence of heparin or of a specific antibody directed against the rAAV capsid, enabling effective therapeutic delivery of a chondrogenic sox9 sequence leading to an enhanced chondrocyte differentiation of the cells. The present findings highlight the value of PEO-PPO copolymers as powerful tools for rAAV-based cartilage regenerative medicine. STATEMENT OF SIGNIFICANCE While recombinant adeno-associated viral (rAAV) vectors are adapted vectors to treat a variety of human disorders, their clinical use is still restricted by pre-existing antiviral immune responses, by a low efficacy of natural vector entry in the target cells, and by inhibition of viral uptake by clinically used compounds like heparin. The search for alternative routes of rAAV delivery is thus becoming a new field of investigation. In the present study, we describe the strong benefits of providing rAAV to human mesenchymal stem cells, a potent source of cells for regenerative medicine, encapsulated in polymeric micelles based on poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) triblock copolymers as novel, effective and safe delivery systems for human gene therapy.

TGF‐β gene transfer and overexpression via rAAV vectors stimulates chondrogenic events in human bone marrow aspirates

Genetic modification of marrow concentrates may provide convenient approaches to enhance the chondrogenic differentiation processes and improve the repair capacities in sites of cartilage defects following administration in the lesions. Here, we provided clinically adapted recombinant adeno‐associated virus (rAAV) vectors to human bone marrow aspirates to promote the expression of the potent transforming growth factor beta (TGF‐β) as a means to regulate the biological and chondrogenic activities in the samples in vitro. Successful TGF‐β gene transfer and expression via rAAV was reached relative to control (lacZ) treatment (from 511.1 to 16.1 pg rhTGF‐β/mg total proteins after 21 days), allowing to durably enhance the levels of cell proliferation, matrix synthesis, and chondrogenic differentiation. Strikingly, in the conditions applied here, application of the candidate TGF‐β vector was also capable of reducing the hypertrophic and osteogenic differentiation processes in the aspirates, showing the potential benefits of using this particular vector to directly modify marrow concentrates to generate single‐step, effective approaches that aim at improving articular cartilage repair in vivo.

rAAV-mediated combined gene transfer and overexpression of TGF-β and SOX9 remodels human osteoarthritic articular cartilage.

Direct administration of therapeutic candidate gene sequences using the safe and effective recombinant adeno‐associated virus (rAAV) vectors is a promising strategy to stimulate the biologic activities of articular chondrocytes as an adapted tool to treat human osteoarthritic (OA) cartilage. In the present study, we developed a combined gene transfer approach based on the co‐delivery of the pleiotropic transformation growth factor beta (TGF‐β) with the specific transcription factor SOX9 via rAAV to human normal and OA chondrocytes in vitro and cartilage explants in situ in light of the mitogenic and pro‐anabolic properties of these factors. Effective, durable co‐overexpression of TGF‐β and SOX9 significantly enhanced the levels of cell proliferation both in human normal and OA chondrocytes and cartilage explants over an extended period of time (21 days), while stimulating the biosynthesis of key matrix components (proteoglycans, type‐II collagen) compared with control conditions (reporter lacZ gene transfer, absence of vector treatment). Of further note, expression of hypertrophic type‐X collagen significantly decreased following co‐treatment by the candidate vectors. The present findings show the value of combining the transfer and expression of potent candidate factors in human OA cartilage as a means to re‐establish essential features of normal cartilage and counteract the pathological shift of homeostasis. These observations support the concept of developing dual therapeutic rAAV gene transfer strategies as future, adapted tools for the direct treatment of human OA.

PEO-PPO-PEO Carriers for rAAV-Mediated Transduction of Human Articular Chondrocytes in Vitro and in a Human Osteochondral Defect Model

Gene therapy is an attractive strategy for the durable treatment of human osteoarthritis (OA), a gradual, irreversible joint disease. Gene carriers based on the small human adeno-associated virus (AAV) exhibit major efficacy in modifying damaged human articular cartilage in situ over extended periods of time. Yet, clinical application of recombinant AAV (rAAV) vectors remains complicated by the presence of neutralizing antibodies against viral capsid elements in a majority of patients. The goal of this study was to evaluate the feasibility of delivering rAAV vectors to human OA chondrocytes in vitro and in an experimental model of osteochondral defect via polymeric micelles to protect gene transfer from experimental neutralization. Interaction of rAAV with micelles of linear (poloxamer PF68) or X-shaped (poloxamine T908) poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) copolymers (PEO-PPO-PEO micelles) was characterized by means of isothermal titration calorimetry. Micelle encapsulation allowed an increase in both the stability and bioactivity of rAAV vectors and promoted higher levels of safe transgene (lacZ) expression both in vitro and in experimental osteochondral defects compared with that of free vector treatment without detrimental effects on the biological activity of the cells or their phenotype. Remarkably, protection against antibody neutralization was also afforded when delivering rAAV via PEO-PPO-PEO micelles in all systems evaluated, especially when using T908. Altogether, these findings show the potential of PEO-PPO-PEO micelles as effective tools to improve current gene-based treatments for human OA.

Overexpression of TGF-β via rAAV-Mediated Gene Transfer Promotes the Healing of Human Meniscal Lesions Ex Vivo on Explanted Menisci

Background: Direct application of therapeutic gene vectors derived from the adeno-associated virus (AAV) might be beneficial to improve the healing of meniscal tears. Purpose: To test the ability of recombinant AAV (rAAV) to overexpress the potent transforming growth factor–β (TGF-β) in primary cultures of human meniscal fibrochondrocytes, in human meniscal explants, and in experimental human meniscal lesions as a new tool to enhance meniscal repair. Study Design: Controlled laboratory study. Methods: The effects of the candidate treatment on the proliferative and metabolic activities of meniscal cells were monitored in vitro for up to 21 days and in situ in intact and injured human menisci for up to 15 days using biochemical, immunohistochemical, histological, and histomorphometric analyses. Results: Efficient production of TGF-β via rAAV was achieved in vitro and in situ, both in the intact and injured meniscus. Application of the rAAV TGF-β vector stimulated the levels of cell proliferation and matrix synthesis (type I collagen) compared with control gene transfer in all systems tested, especially in situ in regions of poor healing capacity and in sites of meniscal injury. No adverse effects of the candidate treatment were observed at the level of osseous differentiation, as tested by immunodetection of type X collagen. Most remarkably, a significant reduction of the amplitude of meniscal tears was noted after TGF-β treatment, an effect that was associated with increased expression levels of the α–smooth muscle actin contractile marker. Conclusion: Overexpression of TGF-β via rAAV gene transfer is capable of modulating the reparative activities of human meniscal cells, allowing for the healing of meniscal lesions by convenient injection in sites of injury. Clinical Relevance: Direct gene-based approaches using rAAV have strong potential to develop new therapeutic options that aim at treating human meniscal defects.

Genetic Modification of Human Peripheral Blood Aspirates Using Recombinant Adeno‐Associated Viral Vectors for Articular Cartilage Repair with a Focus on Chondrogenic Transforming Growth Factor‐β Gene Delivery

Transplantation of genetically modified peripheral blood aspirates that carry chondrogenically competent progenitor cells may offer new, convenient tools to treat articular cartilage lesions compared with the more complex and invasive application of bone marrow concentrates or of bone marrow‐derived mesenchymal stem cells. Here, we show that recombinant adeno‐associated viral (rAAV) vectors are powerful gene vehicles capable of successfully targeting primary human peripheral blood aspirates in a stable and safe manner, allowing for an efficient and long‐term transgene expression in such samples (up to 63 days with use of a lacZ reporter gene and for at least 21 days with application of the pleiotropic, chondrogenic factor transforming growth factor‐β [TGF‐β]). rAAV‐mediated overexpression of TGF‐β enhanced both the proliferative and metabolic properties of the peripheral blood aspirates, also increasing the chondrogenic differentiation processes in these samples. Hypertrophy and osteogenic differentiation events were also activated by production of TGF‐β via rAAV, suggesting that translation of the current approach in vivo will probably require close regulation of expression of this candidate gene. However, these results support the concept of directly modifying peripheral blood as a novel approach to conveniently treat articular cartilage lesions in patients. Stem Cells Translational Medicine 2017;6:249–260