Combined rAAV-based gene therapy and tissue engineering approaches to enhance the molecular mechanisms of articular cartilage repair

Articular cartilage defects do not regenerate. Scaffold-assisted gene therapy is a highly promising novel approach for cartilage repair. Carbon dots, a class of carbon-dominated nanomaterials, are spherical carbonaceous nanomaterials of small sizes exhibiting water dispersibility, chemical stability, photoluminescence properties, and photostability. Here, the potential benefits of using carbon dots to deliver genes coding for the chondroreparative sex-determining region Y-type high-mobility group box 9 (SOX9) transcription factor and transforming growth factor beta (TGF-beta via the clinically adapted recombinant adeno-associated virus (rAAV) vectors were investigated as a means to stimulate chondrogenic processes in human bone marrow-derived mesenchymal stromal cells (hMSCs) versus control (reporter rAAV-lacZ vector) application. hMSCs naturally repopulate cartilage defects but tend to lose their chondrogenic potential over time. Carbon dot-guided genetic modification of hMSCs may rejuvenate these cells in the defects either upon administration of carbon dot/rAAV composites in vivo or by implantation of hMSCs genetically modified by the composites. Four carbon dots were tested to identify an optimal compound (CD-1: citric acid, pentaethylenehexamine; CD-2: citric acid, poly(ethylene glycol) (PEG) monomethyl ether MW 550 Da, N,N-dimethylethylenediamine; CD-3: citric acid, branched poly(ethylenimine) MW 600 Da, PEG monomethyl ether MW 2 kDa; CD-4: citric acid, branched poly(ethylenimine) MW 600 Da). All were capable of formulating and releasing rAAV-lacZ for an effective modification of hMSCs. Among them, CD-2 was optimal to effectively and safely deliver rAAV for at least 10 days, the longest time point examined. Administration of therapeutic (SOX9, TGF-beta) rAAV vectors via CD-2 led to an effective overexpression of these genes in hMSCs, enhancing cell proliferation (TGF-beta) and matrix deposition (glycosaminoglycans, type-II collagen) (SOX9, TGF-beta) for at least 21 days relative to control treatments (CD-2 formulating rAAV-lacZ or lacking rAAV), while restricting undesirable type-I and -X collagen deposition (SOX9, TGF-beta). These results show the potential of carbon dot-guided rAAV modification of hMSCs as a minimally invasive system for translational strategies aiming at enhancing cartilage repair.Defekte des hyalinen Gelenkknorpels regenerieren nicht. Die gerüstgestützte Gentherapie ist ein vielversprechender neuer Ansatz für die Knorpelreparatur. Carbon-Dots (Kohlenstoff-Punkte), eine Klasse kohlenstoffdominierter Nanomaterialien, sind kugelförmige kohlenstoffhaltige Nanomaterialien kleiner Größe, die Wasserdispergierbarkeit, chemische Stabilität, Photolumineszenz sowie und Photostabilität aufweisen. In der vorliegenden Arbeit wurden die potenziellen Vorteile der Verwendung von Carbon-Dots zur Bereitstellung von Genvektoren auf Basis der klinisch-adaptiven rekombinanten adeno-assoziierten viralen (rAAV) Vektoren für den chondrogenen Transkriptionsfaktor SOX9 (geschlechtsbestimmende Region Y-Typ-Hochmobilität-sgruppenbox 9) und den transformierenden Wachstumsfaktor beta (TGF-) zur Stimulierung chondrogener Prozesse in aus humanem Knochenmark gewonnenen mesenchymalen Stromazellen (hMSCs) im Vergleich zur Kontrollanwendung (Reporter-rAAV-lacZ-Vektor) untersucht. Obwohl hMSCs auf natürliche Weise Knorpeldefekte besiedeln, neigen sie dazu, ihr chondrogenes Potenzial im Laufe der Zeit zu verlieren. Eine durch Carbon-Dots gesteuerte genetische Modifikation von hMSCs kann diese Zellen in den Defekten entweder durch die Verabreichung von Carbon-Dots/rAAV-Kompositen in vivo oder durch Implantation von durch diese Komposite genetisch modifizierter hMSCs phänotypisch modulieren. Vier Arten von Carbon-Dots wurden analysiert, um eine optimale Verbindung zu identifizieren [CD-1: Zitronensäure, Pentaethylenhexamin; CD-2: Zitronensäure, Polyethylenglykolmonomethylether (Molekülmasse M = 550 Da), N, N-Dimethylethylendiamin; CD-3: Zitronensäure, verzweigtes Polyethylenimin, (M = 600 Da), Polyethylenglykolmonomethylether (M = 2000 Da); CD-4: Zitronensäure, verzweigtes Polyethylenimin (M = 600 Da)]. Alle Komponenten waren in der Lage, rAAV-lacZ für eine wirksame genetische Modifikation von hMSCs zu formulieren und freizusetzen. Unter diesen war CD-2 optimal, um rAAV für mindestens 10 Tage, den längsten untersuchten Zeitpunkt, effektiv und sicher freizusetzen. Die CD-2-vermittelte 7 Verabreichung der therapeutischen SOX9- oder TGF--rAAV-Vektoren führte zu einer wirksamen Überexpression dieser Gene in hMSCs, wodurch die Zellproliferation (TGF-) und die Matrixdeposition (Glykosaminoglykane, Typ-II-Kollagen) (jeweils nach SOX9 und TGF--Therapie) für mindestens 21 Tage im Vergleich zu Kontrollbehandlungen (CD-2, ohne oder mit rAAV-lacZ formuliert) verstärkt wurde, während eine unerwünschte Typ-I- und Typ-X-Kollagenproduktion jeweils nach SOX9 und TGF--Therapie reduziert ist. Diese Ergebnisse zeigen das Potenzial einer durch Carbon-Dots gesteuerten rAAV-Modifikation von hMSCs als minimalinvasives System für translationale Strategien zur Verbesserung der klinischen Knorpelreparatur

rAAV-Mediated Overexpression of SOX9 and TGF-β 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-β), 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-β) rAAV vectors in hMSCs via CD-2 led to the effective overexpression of each independent transgene, promoting enhanced cell proliferation (TGF-β) 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

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

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

Crude Oil Source Identification of Asphalt via ATR-FTIR Approach Combined with Multivariate Statistical Analysis

The types of crude oil for producing asphalt have a decisive influence on various performance measures (including aging resistance and durability) of asphalt. To discriminate and predict the crude oil source of different asphalt samples, a discrimination model was established using 12 greatly different infrared (IR) characteristic absorption peaks (CAPs) as predictive variables. The model was established based on diverse fingerprint recognition technologies (such as principal component analysis (PCA) and multivariate logistic regression analysis) by using attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR). In this way, the crude oil source of different asphalt samples can be effectively discriminated. At first, by using PCA, the 12 CAPs in the IR spectra of asphalt samples were subjected to dimension reduction processing to control the variables of key factors. Moreover, the scores of various principal components in asphalt samples were calculated. Afterwards, the scores of principal components were analysed through modelling based on multivariate logistic regression analysis to discriminate and predict the crude oil source of different asphalt samples. The result showed that the logistic regression model shows a favourable goodness of fit, with the prediction accuracy reaching 93.9% for the crude oil source of asphalt samples. The method exhibits some outstanding advantages (including ease of operation and high accuracy), which is important when controlling the source and quality and improving the performance of asphalt

MicroRNA-26a negatively regulates toll-like receptor 3 expression of rat macrophages and ameliorates pristane induced arthritis in rats

INTRODUCTION: Abnormal toll-like receptor (TLR)3 signaling plays an indispensable role in pathogenesis of both experimental and human rheumatoid arthritis, and microRNAs (miRNAs) might orchestrate this signaling pathway. This study was performed to determine the relationship between miR-26a and TLR3 in rat macrophages and to observe effects of miR-26a mimic on pristane induced arthritis (PIA) in rats. METHODS: Dual luciferase reporter assay was used to validate the direct interaction between miR-26a (a candidate miRNA to target tlr3 mRNA) and tlr3 3′UTR. MiR-26a regulation on TLR3 gene expression was determined using RT-qPCR and Western blotting after miR-26a mimics and inhibitors were transfected into rat macrophage line NR8383 cells. Poly I:C (TLR3 ligand) was used to trigger TLR3 activation, and mRNA expression of its downstream cytokines interferon (ifn)-β and tumor necrosis factor (tnf)-α was accordingly detected to determine the regulation of TLR3 signaling. Expressions of TLR3 and miR-26a were detected during rat bone marrow derived macrophage (BMDM) induction, in pristane stimulated NR8383 cells and spleens from methotrexate (MTX) treated PIA rats. A miR-26a mimic was administrated intraperitoneally to PIA rats, and arthritis severity was evaluated by macroscopic or microscopic observations. RESULTS: Direct target relationship between miR-26a and tlr3 mRNA in rats was confirmed. Modifications of miR-26a function by transfection of miR-26a mimics and inhibitors exhibited corresponding repression and augmentation of TLR3 and its signaling downstream cytokine expressions in NR8383 cells. The alteration of miR-26a expression was negatively related with TLR3 expression during BMDM induction, in pristane-primed NR8383 cells and PIA rat spleens. Moreover, both abnormal expressions were rescued in MTX treated arthritis rat spleens. The miR-26a mimic treatment displayed the depression of TLR3 expression and ameliorated the disease severity in the rats with pristane induced arthritis. CONCLUSIONS: MiR-26a negatively regulates TLR3 signaling via targeting of TLR3 itself in rat macrophages, and this finding provides a novel insight into abnormal TLR3 overexpression during experimental arthritis

44° Convegno Nazionale Associazione Italiana di Acustica

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-associated mortality worldwide with an overall five-year survival rate less than 7%. Accumulating evidence has revealed the cancer preventive and therapeutic effects of metformin, one of the most widely prescribed medications for type 2 diabetes mellitus. However, its role in pancreatic cancer is not fully elucidated. Herein, we aimed to further study the preventive and therapeutic effects of metformin in genetically engineered mouse models of pancreatic cancer. Methods LSL-KrasG12D/+; Pdx1-Cre (KC) mouse model was established to investigate the effect of metformin in pancreatic tumorigenesis suppression; LSL-KrasG12D/+; Trp53fl/+; Pdx1-Cre (KPC) mouse model was used to evaluate the therapeutic efficiency of metformin in PDAC. Chronic pancreatitis was induced in KC mice by peritoneal injection of cerulein. Results Following metformin treatment, pancreatic acinar-to-ductal metaplasia (ADM) and mouse pancreatic intraepithelial neoplasia (mPanIN) were decreased in KC mice. Chronic pancreatitis induced a stroma-rich and duct-like structure and increased the formation of ADM and mPanIN lesions, in line with an increased cytokeratin 19 (CK19)-stained area. Metformin treatment diminished chronic pancreatitis-mediated ADM and mPanIN formation. In addition, it alleviated the percent area of Masson’s trichrome staining, and decreased the number of Ki67-positive cells. In KPC mice, metformin inhibited tumor growth and the incidence of abdominal invasion. More importantly, it prolonged the overall survival. Conclusions Metformin inhibited pancreatic cancer initiation, suppressed chronic pancreatitis-induced tumorigenesis, and showed promising therapeutic effect in PDAC