Effekt der Überexpression des chondrogenen Transkriptionsfaktors SOX9 durch rekombinante adenoassoziierte virale (rAAV) Vektoren in Kombination mit Trägermaterialien auf die frühe Knorpelreparatur im translationalen Großtiermodell

Die Regenerationsfähigkeit des hyalinen Gelenkknorpels ist begrenzt. Die Suche nach innovativen Methoden zur Knorpelheilung ist Gegenstand intensiver Forschung. Gentherapie und Gewebszüchtung (Tissue Engineering) sind vielversprechende Ansätze. Die vorliegende Arbeit untersucht, ob die frühe Reparatur fokaler vollschichtiger Knorpeldefekte im Großtiermodell in vivo durch in situ Applikation von Biomaterialien und eines replikationsdefekten, rekombinanten adeno-assoziierten viralen Genvektors (rAAV), der eine cDNS des humanen Transkriptionsfaktors SOX9 trägt, verbessert wird. Folgende Hypothesen wurden speziell überprüft: (1) Alleinige Überexpression von SOX9 beladenen rAAV-Vektoren verbessert die Knorpelreparatur im Vergleich zur Kontrollgruppe mit lacZ-beladenen rAAV-Vektoren. (2) Überexpression von rAAV-SOX9 in autologem Knochenmarkaspirat verbessert die Knorpelreparatur im Vergleich mit der rAAV-lacZ-Kontrollgruppe. (3) Überexpression von rAAV-SOX9 in Kombination mit einem soliden Biomaterial verbessert die Knorpelreparatur im Vergleich mit der rAAV-lacZ-Kontrollgruppe. (4) Überexpression von rAAV-SOX9 in Kombination mit einem thermosensitiven Hydrogel verbessert die Knorpelreparatur im Vergleich mit der rAAV-lacZ-Kontrollgruppe. (5) Überexpression von rAAV-SOX9 in Kombination mit einem thermosensitiven Hydrogel ist allen anderen hier untersuchten Ansätzen hinsichtlich der Verbesserung der Knorpelreparatur überlegen. Für die Studie wurden zwölf adulte weibliche Göttinger Minipigs ausgewählt und vollschichtige chondrale Defekte in der Trochlea femoris mit entweder rAAV-SOX9, rAAV-SOX9 in Knochenmarkaspirat, einem PCL-Trägermaterial und rAAV-SOX9 in Knochenmarkaspirat, rAAV-SOX9 im thermosensitiven PF127-Hydrogel oder als Negativkontrolle rAAV-lacZ behandelt. Vier Wochen postoperativ erfolgte die Entnahme der Kniegelenke und die Analyse der Defektreparatur makroskopisch, histologisch, immunhistochemisch und in der Mikrofokuscomputertomographie (μ- CT). Die Daten zeigen, dass (1) die alleinige Überexpression von rAAV-SOX9 die Knorpelreparatur im Vergleich mit der rAAV-lacZ-Kontrollgruppe signifikant verbessert. Sie zeigen weiterhin, dass (2) Überexpression von rAAV-SOX9 in autologem Knochenmarkaspirat die Knorpelreparatur im Vergleich mit der rAAVlacZ- Kontrollgruppe verbessert. Die Hypothese, dass (3) eine Überexpression von rAAV-SOX9 in Kombination mit einem soliden Biomaterial die Knorpelreparatur im Vergleich mit der rAAV-lacZ-Kontrollgruppe verbessert, wurde widerlegt. Schließlich demonstrieren die Daten, dass (4) die Überexpression von rAAV-SOX9 in Kombination mit einem thermosensitiven Hydrogel den singulären Parameter „Defektarchitektur“ im Vergleich zur rAAV-lacZ-Kontrollgruppe verbessert. Zuletzt wurde die Hypothese (5) verworfen, dass Überexpression von rAAV-SOX9 in Kombination mit einem thermosensitiven Hydrogel allen anderen untersuchten Ansätzen hinsichtlich der Verbesserung der Knorpelreparatur überlegen ist. Somit zeigen die Daten zusammenfassend, dass die rAAV-vermittelte Überexpression von SOX9 im Tiermodell zu einer verbesserten osteochondralen Reparatur führt. Die Daten demonstrieren weiterhin, dass die Defektreparatur durch Zusatz von Knochenmarkaspirat sowie durch Kombination von Prinzipien der Gentherapie (rAAV-SOX9) mit Tissue Engineering (PF127/PCL) zusätzlich verbessert werden kann.Overexpression of the chondrogenic transcription factor SOX9 via recombinant adeno associated viral (rAAV) vectors in combination with biomaterials and its effect on early cartilage repair in a large animal model The ability of hyaline articular cartilage to regenerate is limited. The quest for innovative methods to treat focal cartilage defects is the subject of intensive research. Gene therapy and tissue engineering are promising approaches in this regard. The present thesis investigates whether the early repair of focal, full-thickness articular cartilage defects in a large animal model in vivo is improved by in situ application of biomaterials and a replication-defective, recombinant adeno associated viral (rAAV) gene vector carrying a cDNA of the human transcription factor SOX9. The following hypotheses were examined: (1) Sole overexpression of rAAV-SOX9 improves cartilage repair as compared to the control group (lacZ-rAAV vectors). (2) Overexpression of rAAV-SOX9 in autologous bone marrow aspirate improves the cartilage repair in comparison with the rAAV-lacZ control group. (3) Overexpression of rAAV-SOX9 in combination with a solid biomaterial improves cartilage repair compared with the rAAV-lacZ control group. (4) Overexpression of rAAV-SOX9 in combination with a thermosensitive hydrogel improves cartilage repair in comparison with the rAAV-lacZ control group. (5) Overexpression of rAAV-SOX9 combined with a thermosensitive hydrogel is superior to any other investigated approaches. Twelve adult female Minipigs received focal full-thickness chondral defects in the trochlea groove and were treated with either rAAV-SOX9, rAAV-SOX9 in bone marrow aspirate, rAAV-SOX9 in a poly-ε-caprolacton(PCL) scaffold and rAAV-SOX9 in the thermosensitive PF127 hydrogel, or similarly with rAAV-lacZ as a negative control. Four weeks postoperatively, osteochondral repair was analyzed macroscopically, histologically, immunohistochemically and using microfocus computer tomography (μ-CT). The data show that (1) sole overexpression of rAAV-SOX9 simultaneously improves cartilage repair in comparison with the rAAV-lacZ control group. They further reveal that (2) overexpression of rAAV-SOX9 in autologous bone marrow aspirate improves chondral repair by comparison with the rAAV-lacZ control group. The hypothesis that (3) overexpression of rAAV-SOX9 combined with a solid scaffold leads to better cartilage repair than the lacZ control group was rejected. After all the data show that (4) the overexpression of rAAV-SOX9 in combination with a thermosensitive hydrogel optimizes the singular parameter "defect architecture" compared to the rAAV-lacZ control group. Finally, the hypothesis was rejected that (5) overexpression of rAAVSOX9 in combination with a thermosensitive hydrogel is superior to all other approaches studied regarding articular cartilage repair. In conclusion the data show that rAAV-mediated overexpression of SOX9 in a translational large animal model improves osteochondral repair. The data also demonstrate that defect repair can be further improved by the addition of bone marrow aspirate and by combining gene therapy (rAAV-SOX9) with tissue engineering principles (PF127/scaffold)

Thermosensitive Hydrogel Based on PEO-PPO-PEO Poloxamers for a Controlled In Situ Release of Recombinant Adeno-Associated Viral Vectors for Effective Gene Therapy of Cartilage Defects

Advanced biomaterial-guided delivery of gene vectors is an emerging and highly attractive therapeutic solution for targeted articular cartilage repair, allowing for a controlled and minimally invasive delivery of gene vectors in a spatiotemporally precise manner, reducing intra-articular vector spread and possible loss of the therapeutic gene product. As far as it is known, the very first successful in vivo application of such a biomaterial-guided delivery of a potent gene vector in an orthotopic large animal model of cartilage damage is reported here. In detail, an injectable and thermosensitive hydrogel based on poly(ethylene oxide) (PEO)-poly(propylene oxide) (PPO)-PEO poloxamers, capable of controlled release of a therapeutic recombinant adeno-associated virus (rAAV) vector overexpressing the chondrogenic sox9 transcription factor in full-thickness chondral defects, is applied in a clinically relevant minipig model in vivo. These comprehensive analyses of the entire osteochondral unit with multiple standardized evaluation methods indicate that rAAV-FLAG-hsox9/PEO-PPO-PEO hydrogel-augmented microfracture significantly improves cartilage repair with a collagen fiber orientation more similar to the normal cartilage and protects the subchondral bone plate from early bone loss

Thermosensitive Hydrogel Based on PEO–PPO–PEO Poloxamers for a Controlled In Situ Release of Recombinant Adeno-Associated Viral Vectors for Effective Gene Therapy of Cartilage Defects

[Abstract] Advanced biomaterial-guided delivery of gene vectors is an emerging and highly attractive therapeutic solution for targeted articular cartilage repair, allowing for a controlled and minimally invasive delivery of gene vectors in a spatiotemporally precise manner, reducing intra-articular vector spread and possible loss of the therapeutic gene product. As far as it is known, the very first successful in vivo application of such a biomaterial-guided delivery of a potent gene vector in an orthotopic large animal model of cartilage damage is reported here. In detail, an injectable and thermosensitive hydrogel based on poly(ethylene oxide) (PEO)–poly(propylene oxide) (PPO)–PEO poloxamers, capable of controlled release of a therapeutic recombinant adeno-associated virus (rAAV) vector overexpressing the chondrogenic sox9 transcription factor in full-thickness chondral defects, is applied in a clinically relevant minipig model in vivo. These comprehensive analyses of the entire osteochondral unit with multiple standardized evaluation methods indicate that rAAV-FLAG-hsox9/PEO–PPO–PEO hydrogel-augmented microfracture significantly improves cartilage repair with a collagen fiber orientation more similar to the normal cartilage and protects the subchondral bone plate from early bone loss.H.M. and L.G. contributed equally to this work. This project wassupported by the Deutsche Forschungsgemeinschaft (DFG RE 3828/2-1). All animal experiments were conducted in agreement with the nationallegislation on protection of animals and the National Institutes ofHealth (NIH) Guidelines for the Care and Use of Laboratory Animals(NIH Publication 85-23, Rev 1985) and were approved by the SaarlandUniversity Animal Committee according to German guidelines.Deutschland. Deutsche Forschungsgemeinschaft; DFG RE 3828/2-

Circulation and Oxygen Distribution in the Tropical Atlantic Cruise No. 80, Leg 1; October 26 to November 23, 2009 Mindelo (Cape Verde) to Mindelo (Cape Verde)

METEOR cruise 80/1 was a contribution to the SFB 754 “Climate-Biogeochemistry Interactions in the Tropical Ocean”. Shipboard, glider and moored observations are used to study the temporal and spatial variability of physical and biogeochemical parameters within the oxygen minimum zone (OMZ) of the tropical North Atlantic. As part of the BMBF “Nordatlantik” project, it further focuses on the equatorial current system including the Equatorial Undercurrent (EUC) and intermediate currents below. During the cruise, hydrographic station observations were performed using a CTD/O2 rosette, including water sampling for salinity, oxygen, nutrients and other biogeochemical tracers. Underway current measurements were successfully carried out with the 75 kHz ADCP borrowed from R/V POSEIDON during the first part of the cruise, and R/V METEOR’s 38 kHz ADCP during the second part. During M80/1, an intensive mooring program was carried out with 8 mooring recoveries and 8 mooring deployments. Right at the beginning of the cruise, a multidisciplinary mooring near the Cape Verde Islands was recovered and redeployed. Within the framework of SFB 754, two moorings with CTD/O2 profilers were recovered and redeployed with other instrumentation in the center and at the southern rim of the OMZ of the tropical North Atlantic. The equatorial mooring array as part of BMBF “North Atlantic” project consists of 5 current meter moorings along 23°W between 2°S and 2°N. It is aimed at quantifying the variability of the thermocline water supply toward the equatorial cold tongue which develops east of 10°W during boreal summer. Several glider missions were performed during the cruise. One glider was recovered that was deployed two months earlier. Another glider was deployed for two short term missions, near the equator for about 8 days and near 8°N for one day. This glider was equipped with a new microstructure probe in addition to standard sensors, i.e. CTD/O2, chlorophyll and turbidity

Prevalence of mass social media-induced illness presenting with Tourette-like behavior in Germany between 2019 and 2021

Starting in 2019, in Germany the first well documented outbreak of mass sociogenic illness induced by social media (mass social media-induced illness; MSMI) occurred presenting with functional Tourette-like behaviors (FTB). This study aimed to provide first data on the prevalence rate of MSMI-FTB in Germany between 2019 and 2021 in the general population. We conducted a large-scale representative population survey in cooperation with the USUMA market and social research institute. Between August and December 2021, n = 2.509 people (mean age: 49.5 years, range: 16–95 years, n = 1.276 females) were randomly selected, visited in their households, interviewed, and asked to answer for themselves, but also for close family members (n = 6.744). Thus, in total, we received answers for n = 9.253 people. Probable MSMI-FTB was found in n = 33 individuals (mean age at onset: 30.5 years, n = 8 females). Based on strict criteria, the diagnosis of MSMI-FTB was considered highly likely in 16/33 individuals (mean age at onset: 25.6 years, n = 2 females) corresponding to prevalence rates of 0.17% (CIlower = 0.10, CIupper = 0.28) and 0.36% (CIlower = 0.25, CIupper = 0.50), respectively. This is the first large-scale, population representative study investigating the prevalence of MSMI-FTB in the general population in Germany between 2019 and 2021. Based on the prevalence rates found, MSMI-FTB is highly relevant for health economy. Accordingly, we suggest educating healthcare professionals and the general public to avoid misdiagnosis and inefficient treatment.<br/

Association of FKBP5 genotype with depressive symptoms in patients with coronary heart disease: a prospective study

Depression and coronary heart disease (CHD) are prevalent and often co-occurring disorders. Both have been associated with a dysregulated stress system. As a central element of the stress system, the FKBP5 gene has been shown to be associated with depression. In a prospective design, this study aims to investigate the association of FKBP5 with depressive symptoms in CHD patients. N = 268 hospitalized CHD patients were included. Depressive symptoms were measured using the Hospital Anxiety and Depression Scale (HADS-D) at four time points (baseline, and after 1 month, 6 months, and 12 months). The functional FKBP5 single-nucleotide polymorphism (SNP) rs1360780 was selected for genotyping. Linear regression models showed that a higher number of FKBP5 C alleles was associated with more depressive symptoms in CHD patients both at baseline (p = 0.015) and at 12-months follow-up (p = 0.025) after adjustment for confounders. Further analyses revealed that this effect was driven by an interaction of FKBP5 genotype with patients' prior CHD course. Specifically, only in patients with a prior myocardial infarction or coronary revascularization, more depressive symptoms were associated with a higher number of C alleles (baseline: p = 0.046; 1-month: p = 0.026; 6-months: p = 0.028). Moreover, a higher number of C alleles was significantly related to a greater risk for dyslipidemia (p = .016). Our results point to a relevance of FKBP5 in the association of the two stress-related diseases depression and CHD