The genetic modification of freshly aspirated bone marrow may provide convenient tools to enhance the regenerative capacities of cartilage defects compared with the complex manipulation of isolated progenitor cells. In the present study, we examined the ability and safety of recombinant adeno-associated virus (rAAV) serotype 2 vectors to deliver various reporter gene sequences in primary human bone marrow aspirates over time without altering the chondrogenic processes in the samples. The results demonstrate that successful rAAV-mediated gene transfer and expression of the lacZ and red fluorescent protein marker genes were achieved in transduced aspirates at very high efficiencies (90-94%) and over extended periods of time (up to 125 days) upon treatment with hirudin, an alternative anticoagulant that does not prevent the adsorption of the rAAV-2 particles at the surface of their targets compared with heparin. Application of rAAV was safe, displaying neither cytotoxic nor detrimental effects on the cellular and proliferative activities or on the chondrogenic processes in the aspirates especially using an optimal dose of 0.5 mg ml(-1) hirudin, and application of the potent SOX9 transcription factor even enhanced these processes while counteracting hypertrophic differentiation. The current findings demonstrate the clinical value of this class of vector to durably and safely modify bone marrow aspirates as a means to further develop convenient therapeutic approaches to improve the healing of cartilage defects.

Introduction: The transplantation of genetically modified progenitor cells such as bone marrow-derived mesenchymal stem cells (MSCs) is an attractive strategy to improve the natural healing of articular cartilage defects. In the present study, we examined the potential benefits of sustained overexpression of the mitogenic and pro-anabolic insulin-like growth factor I (IGF-I) via gene transfer upon the biological activities of human MSCs (hMSCs).

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 ex-

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.

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Direct therapeutic gene transfer in marrow concentrates is an attractive strategy to conveniently enhance the chondrogenic differentiation processes as a means to improve the healing response of damaged articular cartilage upon reimplantation in sites of injury. In the present study, we evaluated the ability of the clinically adapted recombinant adeno-associated virus (rAAV) vectors to mediate overexpression of the insulin-like growth factor I (IGF-I) in human bone marrow aspirates that may modulate the proliferative, anabolic activities, and chondrogenic differentiation potential in such samples in vitro. The results demonstrate that successful, significant rAAV-mediated IGF-I gene transfer and expression were achieved in transduced aspirates (up to 105.9 -35.1 pg rhIGF-I/mg total proteins) over time (21 days) at very high levels (*80% of cells expressing the candidate IGF-I transgene), leading to increased levels of proliferation, matrix synthesis, and chondrogenic differentiation over time compared with the control (lacZ) condition. Treatment with the candidate IGF-I vector also stimulated the hypertrophic and osteogenic differentiation processes in the aspirates, suggesting that the regulation of IGF-I expression through rAAV will be a prerequisite for future translation of the approach in vivo. However, these findings show the possible benefits of this vector class to directly modify marrow concentrates as a convenient tool for strategies that aim at improving the repair of articular cartilage lesions.

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.

Immune checkpoint inhibitors (ICIs) can block distinct receptors on T cells or tumor cells thus preventing T cell inactivation and tumor immune escape. While the clinical response to treatment with ICIs in cancer patients is impressive, this therapy is often associated with a number of immune-related adverse events. There is therefore a need to explore innovative strategies of tumor-specific delivery of ICIs. Delivery of therapeutic proteins on a genetic level can be accomplished with viral vectors including those derived from adeno-associated virus (AAV). Here, we assessed the tumor-targeted Her2-AAV, a receptor-targeted AAV vector binding to the tumor antigen Her2/neu for cell entry, as vehicle for ICI gene delivery. Initially, we packaged the coding sequence of a scFv-Fc fusion protein directed against mouse programmed cell death protein-1 (PD-1) into Her2-AAV. Upon transduction of Her2/neu + RENCA cells, AAV-encoded αPD-1 was readily detectable in the cell culture supernatant and revealed specific binding to its target antigen. In vivo , in BALB/c mice bearing subcutaneous RENCA-Her2/neu tumors, Her2-AAV mediated specific gene delivery into tumor tissue upon intravenous administration as verified by luciferase gene transfer and in vivo imaging thus demonstrating unimpaired tumor-targeting by Her2-AAV vectors in immunocompetent animals. When delivering the αPD-1 gene, levels of ICI were similar in tumor tissue for Her2-AAV and AAV2 but substantially reduced in liver for Her2-AAV. When combined with chemotherapy a tendency for reduced progression of tumor growth was documented for Her2-AAV treated mice. To get closer to the clinical situation, AAV constructs that deliver the complete coding sequence of the therapeutic antibody nivolumab which is directed against human PD-1 were generated next. The AAV-Nivolumab constructs were expressed and released from transduced MDA-MB-453 cells in vitro and from RENCA-Her2/neu cells upon intratumoral as well as intravenous administration in vivo . Antibody processing and expression levels were further improved through optimization of construct design. In conclusion, we provide proof-of-principle for redirecting the biodistribution of ICIs from liver and serum to tumor tissue by the use of engineered AAV vectors. This strategy can be easily combined with other types of immunotherapeutic concepts.