Cardiomyopathy in chronic kidney disease: clinical features, biomarkers and the contribution of murine models in understanding pathophysiology.

The cardiorenal syndrome (CRS) is described as a multi-organ disease encompassing bidirectionally heart and kidney. In CRS type 4, chronic kidney disease (CKD) leads to cardiac injury. Different pathological mechanisms have been identified to contribute to the establishment of CKD-induced cardiomyopathy, including a neurohormonal dysregulation, disturbances in the mineral metabolism and an accumulation of uremic toxins, playing an important role in the development of inflammation and oxidative stress. Combined, this leads to cardiac dysfunction and cardiac pathophysiological and morphological changes, like left ventricular hypertrophy, myocardial fibrosis and cardiac electrical changes. Given that around 80% of dialysis patients suffer from uremic cardiomyopathy, the study of cardiac outcomes in CKD is clinically highly relevant. The present review summarizes clinical features and biomarkers of CKD-induced cardiomyopathy and discusses underlying pathophysiological mechanisms recently uncovered in the literature. It discloses how animal models have contributed to the understanding of pathological kidney-heart crosstalk, but also provides insights into the variability in observed effects of CKD on the heart in different CKD mouse models, covering both "single hit" as well as "multifactorial hit" models. Overall, this review aims to support research progress in the field of CKD-induced cardiomyopathy.

Pro-oxidative priming but maintained cardiac function in a broad spectrum of murine models of chronic kidney disease.

AIMS: Patients with chronic kidney disease (CKD) have an increased risk of cardiovascular events and exhibit myocardial changes including left ventricular (LV) hypertrophy and fibrosis, overall referred to as 'uremic cardiomyopathy'. Although different CKD animal models have been studied for cardiac effects, lack of consistent reporting on cardiac function and pathology complicates clear comparison of these models. Therefore, this study aimed at a systematic and comprehensive comparison of cardiac function and cardiac pathophysiological characteristics in eight different CKD models and mouse strains, with a main focus on adenine-induced CKD. METHODS AND RESULTS: CKD of different severity and duration was induced by subtotal nephrectomy or adenine-rich diet in various strains (C57BL/6J, C57BL/6 N, hyperlipidemic C57BL/6J ApoE-/-, 129/Sv), followed by the analysis of kidney function and morphology, blood pressure, cardiac function, cardiac hypertrophy, fibrosis, myocardial calcification and inflammation using functional, histological and molecular techniques, including cardiac gene expression profiling supplemented by oxidative stress analysis. Intriguingly, despite uremia of variable degree, neither cardiac dysfunction, hypertrophy nor interstitial fibrosis were observed. However, already moderate CKD altered cardiac oxidative stress responses and enhanced oxidative stress markers in each mouse strain, with cardiac RNA sequencing revealing activation of oxidative stress signaling as well as anti-inflammatory feedback responses. CONCLUSION: This study considerably expands the knowledge on strain- and protocol-specific differences in the field of cardiorenal research and reveals that several weeks of at least moderate experimental CKD increase oxidative stress responses in the heart in a broad spectrum of mouse models. However, this was insufficient to induce relevant systolic or diastolic dysfunction, suggesting that additional "hits" are required to induce uremic cardiomyopathy. TRANSLATIONAL PERSPECTIVE: Patients with chronic kidney disease (CKD) have an increased risk of cardiovascular adverse events and exhibit myocardial changes, overall referred to as 'uremic cardiomyopathy'. We revealed that CKD increases cardiac oxidative stress responses in the heart. Nonetheless, several weeks of at least moderate experimental CKD do not necessarily trigger cardiac dysfunction and remodeling, suggesting that additional "hits" are required to induce uremic cardiomyopathy in the clinical setting. Whether the altered cardiac oxidative stress balance in CKD may increase the risk and extent of cardiovascular damage upon additional cardiovascular risk factors and/or events will be addressed in future studies.

A systematic review and meta-analysis of murine models of uremic cardiomyopathy.

Chronic kidney disease (CKD) triggers the risk of developing uremic cardiomyopathy as characterized by cardiac hypertrophy, fibrosis and functional impairment. Traditionally, animal studies are used to reveal the underlying pathological mechanism, although variable CKD models, mouse strains and readouts may reveal diverse results. Here, we systematically reviewed 88 studies and performed meta-analyses of 52 to support finding suitable animal models for future experimental studies on pathological kidney-heart crosstalk during uremic cardiomyopathy. We compared different mouse strains and the direct effect of CKD on cardiac hypertrophy, fibrosis and cardiac function in "single hit" strategies as well as cardiac effects of kidney injury combined with additional cardiovascular risk factors in "multifactorial hit" strategies. In C57BL/6 mice, CKD was associated with a mild increase in cardiac hypertrophy and fibrosis and marginal systolic dysfunction. Studies revealed high variability in results, especially regarding hypertrophy and systolic function. Cardiac hypertrophy in CKD was more consistently observed in 129/Sv mice, which express two instead of one renin gene and more consistently develop increased blood pressure upon CKD induction. Overall, "multifactorial hit" models more consistently induced cardiac hypertrophy and fibrosis compared to "single hit" kidney injury models. Thus, genetic factors and additional cardiovascular risk factors can "prime" for susceptibility to organ damage, with increased blood pressure, cardiac hypertrophy and early cardiac fibrosis more consistently observed in 129/Sv compared to C57BL/6 strains.

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.

STIM-Orai Channels and Reactive Oxygen Species in the Tumor Microenvironment.

The tumor microenvironment (TME) is shaped by cancer and noncancerous cells, the extracellular matrix, soluble factors, and blood vessels. Interactions between the cells, matrix, soluble factors, and blood vessels generate this complex heterogeneous microenvironment. The TME may be metabolically beneficial or unbeneficial for tumor growth, it may favor or not favor a productive immune response against tumor cells, or it may even favor conditions suited to hijacking the immune system for benefitting tumor growth. Soluble factors relevant for TME include oxygen, reactive oxygen species (ROS), ATP, Ca2+, H⁺, growth factors, or cytokines. Ca2+ plays a prominent role in the TME because its concentration is directly linked to cancer cell proliferation, apoptosis, or migration but also to immune cell function. Stromal-interaction molecules (STIM)-activated Orai channels are major Ca2+ entry channels in cancer cells and immune cells, they are upregulated in many tumors, and they are strongly regulated by ROS. Thus, STIM and Orai are interesting candidates to regulate cancer cell fate in the TME. In this review, we summarize the current knowledge about the function of ROS and STIM/Orai in cancer cells; discuss their interdependencies; and propose new hypotheses how TME, ROS, and Orai channels influence each other.

Tumor-Specific Delivery of Immune Checkpoint Inhibitors by Engineered AAV Vectors.

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.

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.

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.

Effects of combined rAAV-mediated TGF-ß and sox9 gene transfer and overexpression on the metabolic and chondrogenic activities in human bone marrow aspirates.

BACKGROUND: Transplantation of genetically modified bone marrow concentrates is an attractive approach to conveniently activate the chondrogenic differentiation processes as a means to improve the intrinsic repair capacities of damaged articular cartilage. METHODS: Human bone marrow aspirates were co-transduced with recombinant adeno-associated virus (rAAV) vectors to overexpress the pleiotropic transformation growth factor beta (TGF-ß) and the cartilage-specific transcription factor sox9 as a means to enhance the chondroreparative processes in conditions of specific lineage differentiation. RESULTS: Successful TGF-ß/sox9 combined gene transfer and overexpression via rAAV was achieved in chondrogenically induced human bone marrow aspirates for up to 21 days, the longest time point evaluated, leading to increased proliferation, matrix synthesis, and chondrogenic differentiation relative to control treatments (reporter lacZ treatment, absence of vector application) especially when co-applying the candidate vectors at the highest vector doses tested. Optimal co-administration of TGF-ß with sox9 also advantageously reduced hypertrophic differentiation in the aspirates. CONCLUSIONS: These findings report the possibility of directly modifying bone marrow aspirates by combined therapeutic gene transfer as a potent and convenient future approach to improve the repair of articular cartilage lesions.

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-ß 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-ß [TGF-ß]). rAAV-mediated overexpression of TGF-ß 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-ß 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.

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.

Effects of rAAV-mediated FGF-2 gene transfer and overexpression upon the chondrogenic differentiation processes in human bone marrow aspirates.

BACKGROUND: Application of genetically modified bone marrow concentrates in articular cartilage lesions is a promising approach to enhance cartilage repair by stimulating the chondrogenic differentiation processes in sites of injury. METHOD: In the present study, we examined the potential benefits of transferring the proliferative and pro-chondrogenic basic fibroblast growth factor (FGF-2) to human bone marrow aspirates in vitro using the clinically adapted recombinant adeno-associated virus (rAAV) vectors to monitor the biological and chondrogenic responses over time to the treatment compared with control (lacZ) gene application. RESULTS: Effective, significant FGF-2 gene transfer and expression via rAAV was established in the aspirates relative to the lacZ condition (from ~ 97 to 36 pg rhFGF-2/mg total proteins over an extended period of 21 days). Administration of the candidate FGF-2 vector led to prolonged increases in cell proliferation, matrix synthesis, and chondrogenesis but also to hypertrophic and terminal differentiation in the aspirates. CONCLUSION: The present evaluation shows the advantages of rAAV-mediated FGF-2 gene transfer to conveniently modify bone marrow concentrates as a future approach to directly treat articular cartilage lesions, provided that expression of the growth factor is tightly regulated to prevent premature hypertrophy in vivo.

Gene- and Stem Cell-Based Approaches to Regulate Hypertrophic Differentiation in Articular Cartilage Disorders.

Hypertrophy is a key component of endochondral ossification, the process controlling skeletal (cartilage, bone) development by differentiation of mesenchymal stem cells. Hypertrophic events also occur on cartilage injury such as during osteoarthritis (OA) and to a certain extent in focal lesions that may lead to OA if let untreated. Strategies based on the delivery on therapeutic genes and progenitor cells (the cells mostly recruited in spontaneous and guided repair) offer potential tools to delay or even prevent such undesirable events in sites of cartilage damage. The goal of this review is to revisit the mechanisms of hypertrophy during skeletal development and diseases and to provide an overview of the most recent advances in gene and stem cell therapy in the field of cartilage repair.

rAAV-mediated combined gene transfer and overexpression of TGF-ß 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-ß) 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-ß 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. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2181-2190, 2016.

Co-overexpression of TGF-ß and SOX9 via rAAV gene transfer modulates the metabolic and chondrogenic activities of human bone marrow-derived mesenchymal stem cells.

BACKGROUND: Articular cartilage has a limited potential for self-healing. Transplantation of genetically modified progenitor cells like bone marrow-derived mesenchymal stem cells (MSCs) is an attractive strategy to improve the intrinsic repair capacities of damaged articular cartilage. METHODS: In this study, we examined the potential benefits of co-overexpressing the pleiotropic transformation growth factor beta (TGF-ß) with the cartilage-specific transcription factor SOX9 via gene transfer with recombinant adeno-associated virus (rAAV) vectors upon the biological activities of human MSCs (hMSCs). Freshly isolated hMSCs were transduced over time with separate rAAV vectors carrying either TGF-ß or sox9 in chondrogenically-induced aggregate cultures to evaluate the efficacy and duration of transgene expression and to monitor the effects of rAAV-mediated genetic modification upon the cellular activities (proliferation, matrix synthesis) and chondrogenic differentiation potency compared with control conditions (lacZ treatment, sequential transductions). RESULTS: Significant, prolonged TGF-ß/sox9 co-overexpression was achieved in chondrogenically-induced hMSCs upon co-transduction via rAAV for up to 21 days, leading to enhanced proliferative, biosynthetic, and chondrogenic activities relative to control treatments, especially when co-applying the candidate vectors at the highest vector doses tested. Optimal co-administration of TGF-ß with sox9 also advantageously reduced hypertrophic differentiation of the cells in the conditions applied here. CONCLUSION: The present findings demonstrate the possibility of modifying MSCs by combined therapeutic gene transfer as potent future strategies for implantation in clinically relevant animal models of cartilage defects in vivo.

TGF-ß 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-ß) as a means to regulate the biological and chondrogenic activities in the samples in vitro. Successful TGF-ß gene transfer and expression via rAAV was reached relative to control (lacZ) treatment (from 511.1 to 16.1 pg rhTGF-ß/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-ß 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.

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

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.

DNA repair by MGMT, but not AAG, causes a threshold in alkylation-induced colorectal carcinogenesis.

Epidemiological studies indicate that N-nitroso compounds (NOC) are causally linked to colorectal cancer (CRC). NOC induce DNA alkylations, including O (6)-methylguanine (O (6)-MeG) and N-methylated purines, which are repaired by O (6)-MeG-DNA methyltransferase (MGMT) and N-alkyladenine-DNA glycosylase (AAG)-initiated base excision repair, respectively. In view of recent evidence of nonlinear mutagenicity for NOC-like compounds, the question arises as to the existence of threshold doses in CRC formation. Here, we set out to determine the impact of DNA repair on the dose-response of alkylation-induced CRC. DNA repair proficient (WT) and deficient (Mgmt (-/-), Aag (-/-) and Mgmt (-/-)/Aag (-/-)) mice were treated with azoxymethane (AOM) and dextran sodium sulfate to trigger CRC. Tumors were quantified by non-invasive mini-endoscopy. A non-linear increase in CRC formation was observed in WT and Aag (-/-) mice. In contrast, a linear dose-dependent increase in tumor frequency was found in Mgmt (-/-) and Mgmt (-/-)/Aag (-/-) mice. The data were corroborated by hockey stick modeling, yielding similar carcinogenic thresholds for WT and Aag (-/-) and no threshold for MGMT lacking mice. O (6)-MeG levels and depletion of MGMT correlated well with the observed dose-response in CRC formation. AOM induced dose-dependently DNA double-strand breaks in colon crypts including Lgr5-positive colon stem cells, which coincided with ATR-Chk1-p53 signaling. Intriguingly, Mgmt (-/-) mice displayed significantly enhanced levels of γ-H2AX, suggesting the usefulness of γ-H2AX as an early genotoxicity marker in the colorectum. This study demonstrates for the first time a non-linear dose-response for alkylation-induced colorectal carcinogenesis and reveals DNA repair by MGMT, but not AAG, as a key node in determining a carcinogenic threshold.

Chondrogenic Differentiation Processes in Human Bone Marrow Aspirates upon rAAV-Mediated Gene Transfer and Overexpression of the Insulin-Like Growth Factor I.

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.