Development of transposon and retrovirus-derived vectors for the rapid establishment of efficient producer cell lines

With the growing demand for therapeutic proteins such as antibodies or vaccines, manufactures are in need to deliver products at high quality and constant productivity. For the biotechnological production of these products, mammalian cells are predominantly used. The productivity of producer cells is, amongst others, dependent on the number of integrated transgene copies per cell, and thus elevating stable gene transfer efficiencies to ensure sustained expression of the gene of interest is paramount. In this study the Sleeping Beauty transposon two-component vector system, consisting of a transposon donor plasmid and a transposase expression plasmid, was used to generate stable cell pools. However, using sensitive genomic PCR and reverse transcriptase PCR (RT-PCR), the transposase gene integration and expression was demonstrated for a time period of 48 days post transfection. To provide an alternative to the employment of plasmid-based transposase expression circumventing potential re-mobilization events of the already stably transposed transgenes, the transposase gene was transcribed in vitro into mRNA. After co-transfection of transposase transcripts at different ratios to the donor vector efficient transposition was obtained. This study demonstrated that this technical approach mediated high copy numbers and expression levels of transgenes in recombinant cells without the risk of undesired extended transposase expression. Besides transposons, retroviral vectors are frequently used to introduce foreign genetic material into mammalian cells aiming for the establishment of producer cells. Such viral vectors are commonly pseudotyped with VSV-G achieving high vector copy numbers in mammalian cells. However, this requires handling under BSL-2 conditions. To circumvent this, viral vectors were equipped with the ecotropic envelope protein PVC211mc, a molecular clone of Friend murine leukemia virus (MLV), enabling transduction of CHO cells and murine hematopoietic stem cells but allowing experiments in BSL- 1 laboratories. The aim of this work was to optimize gene transfer efficiencies by generating PVC211-derived envelope protein (Env) variants lacking the R-peptide and thus rendering the Env proteins fusogenic. All generated variants failed to efficiently pseudotype MLV vectors but two variants successfully pseudotyped HIV-1 particles. The HIV-1 vectors pseudotyped with the envelope variant eMLV-GaLVΔR mediated superior infectivity as compared to wild-type Env. HIV(eMLV-GaLVΔR) should prove useful as a tool for the establishment of productive producer cells.German Federal Ministry of Education and Research/Forschung an Fachhochschulen/13FH242PX6/E

Haushaltskonsolidierung in Kommunen des Landes Sachsen-Anhalt

Das Ziel der vorliegenden Bachelorarbeit besteht in der empirischen Untersuchung der Konsolidierungsbemühungen der Kommunen im Land Sachsen-Anhalt. Dabei steht im Fokus der empirischen Forschung, welche Maßnahmen die Kommunen im Land Sachsen-Anhalt ergreifen, um ihre Haushalte zu konsolidieren und welche Umsetzungserfahrungen sie dabei gemacht haben. Gleichzeitig sollen die Einschätzungen der Kommunen zur zukünftigen Entwicklung untersucht werden

Transgene Expression and Transposition Efficiency of Two-Component Sleeping Beauty Transposon Vector Systems Utilizing Plasmid or mRNA Encoding the Transposase

The use of two-component transposon plasmid vector systems, namely, a transposase construct and a donor vector carrying the gene of interest (GOI) can accelerate the development of recombinant cell lines. However, the undesired stable transfection of the transposase construct and the sustained expression of the enzyme can cause genetic instability due to the re-mobilization of the previously transposed donor vectors. Using a Sleeping Beauty-derived vector system, we established three recombinant cell pools and demonstrate stable integration of the transposase construct and sustained expression of the transposase over a period of 48 days. To provide an alternative approach, transcripts of the transposase gene were generated in vitro and co-transfected with donor vector plasmid at different ratios and mediating high GOI copy number integrations and expression levels. We anticipate that the use of transposase mRNA will foster further improvements in future cell line development processes

Novel suspension retroviral packaging cells generated by transposition using transposase encoding mRNA advance vector yields and enable production in bioreactors

To date, the establishment of high-titer stable viral packaging cells (VPCs) at large scale for gene therapeutic applications is very time- and cost-intensive. Here we report the establishment of three human suspension 293-F-derived ecotropic MLV-based VPCs. The classic stable transfection of an EGFP-expressing transfer vector resulted in a polyclonal VPC pool that facilitated cultivation in shake flasks of 100 mL volumes and yielded high functional titers of more than 1 × 106 transducing units/mL (TU/mL). When the transfer vector was flanked by transposon terminal inverted repeats (TIRs) and upon co-transfection of a plasmid encoding for the transposase, productivities could be slightly elevated to more than 3 × 106 TU/mL. In contrast and using mRNA encoding for the transposase, as a proof of concept, productivities were drastically improved by more than ten-fold exceeding 5 × 107 TU/mL. In addition, these VPC pools were generated within only 3 weeks. The production volume was successfully scaled up to 500 mL employing a stirred-tank bioreactor (STR). We anticipate that the stable transposition of transfer vectors employing transposase transcripts will be of utility for the future establishment of high-yield VPCs producing pseudotype vector particles with a broader host tropism on a large scale

Association of regional socioeconomic deprivation and rurality with global developmental delay in early childhood: Data from mandatory school entry examinations in Germany

Background: From birth to young adulthood, health and development of young people are strongly linked to their living situation, including their family’s socioeconomic position (SEP) and living environment. The impact of regional characteristics on development in early childhood beyond family SEP has been rarely investigated. This study aimed to identify regional predictors of global developmental delay at school entry taking family SEP into consideration. Method: We used representative, population-based data from mandatory school entry examinations of the German federal state of Brandenburg in 2018/2019 with n=22,801 preschool children. By applying binary multilevel models, we hierarchically analyzed the effect of regional deprivation defined by the German Index of Socioeconomic Deprivation (GISD) and rurality operationalized as inverted population density of the children’s school district on global developmental delay (GDD) while adjusting for family SEP (low, medium and high). Results: Family SEP was significantly and strongly linked to GDD. Children with the highest family SEP showed a lower odds for GDD compared to a medium SEP (female: OR=4.26, male: OR=3.46) and low SEP (female: OR=16.58, male: OR=12.79). Furthermore, we discovered a smaller, but additional and independent effect of regional socioeconomic deprivation on GDD, with a higher odds for children from a more deprived school district (female: OR=1.35, male: OR=1.20). However, rurality did not show a significant link to GDD in preschool children beyond family SEP and regional deprivation. Conclusion: Family SEP and regional deprivation are risk factors for child development and of particular interest to promote health of children in early childhood and over the life course.Peer Reviewe

Mood and anxiety disorders within the Research Domain Criteria framework of Positive and Negative Valence Systems: a scoping review

IntroductionWhile a growing body of research is adopting Research Domain Criteria (RDoC)-related methods and constructs, there is still a lack of comprehensive reviews on the state of published research on Positive Valence Systems (PVS) and Negative Valence Systems (NVS) in mood and anxiety disorders consistent with the RDoC framework.MethodsFive electronic databases were searched to identify peer-reviewed publications covering research on “positive valence” and “negative valence” as well as “valence,” “affect,” and “emotion” for individuals with symptoms of mood and anxiety disorders. Data was extracted with a focus on disorder, domain, (sub-) constructs, units of analysis, key results, and study design. Findings are presented along four sections, distinguishing between primary articles and reviews each for PVS, NVS, and cross-domain PVS and NVS.ResultsA total of 231 abstracts were identified, and 43 met the inclusion criteria for this scoping review. Seventeen publications addressed research on PVS, seventeen on NVS, and nine covered cross-domain research on PVS and NVS. Psychological constructs were typically examined across different units of analysis, with the majority of publications incorporating two or more measures. Molecular, genetic, and physiological aspects were mainly investigated via review articles, primary articles focused on self-report, behavioral, and, to a lesser extent, physiological measures.ConclusionsThis present scoping review shows that mood and anxiety disorders were actively studied using a range of genetic, molecular, neuronal, physiological, behavioral, and self-report measures within the RDoC PVS and NVS. Results highlight the essential role of specific cortical frontal brain structures and of subcortical limbic structures in impaired emotional processing in mood and anxiety disorders. Findings also indicate overall limited research on NVS in bipolar disorders and PVS in anxiety disorders, a majority of self-report studies, and predominantly observational studies. Future research is needed to develop more RDoC-consistent advancements and intervention studies targeting neuroscience-driven PVS and NVS constructs

Anxiety in response to the climate and environmental crises: validation of the Hogg Eco-Anxiety Scale in Germany

Background: As the climate and environmental crises unfold, eco-anxiety, defined as anxiety about the crises’ devastating consequences for life on earth, affects mental health worldwide. Despite its importance, research on eco-anxiety is currently limited by a lack of validated assessment instruments available in different languages. Recently, Hogg and colleagues proposed a multidimensional approach to assess eco-anxiety. Here, we aim to translate the original English Hogg Eco-Anxiety Scale (HEAS) into German and to assess its reliability and validity in a German sample. Methods: Following the TRAPD (translation, review, adjudication, pre-test, documentation) approach, we translated the original English scale into German. In total, 486 participants completed the German HEAS. We used Bayesian confirmatory factor analysis (CFA) to assess whether the four-factorial model of the original English version could be replicated in the German sample. Furthermore, associations with a variety of emotional reactions towards the climate crisis, general depression, anxiety, and stress were investigated. Results: The German HEAS was internally consistent (Cronbach’s alphas 0.71–0.86) and the Bayesian CFA showed that model fit was best for the four-factorial model, comparable to the factorial structure of the original English scale (affective symptoms, rumination, behavioral symptoms, anxiety about personal impact). Weak to moderate associations were found with negative emotional reactions towards the climate crisis and with general depression, anxiety, and stress. Discussion: Our results support the original four-factorial model of the scale and indicate that the German HEAS is a reliable and valid scale to assess eco-anxiety in German speaking populations

What is the health status of girls and boys in the COVID-19 pandemic? Selected results of the KIDA study

Background: It is well known that there are gender differences in the health behaviour and physical and mental health of children. The COVID-19 pandemic influenced the health and lifestyles of children and adolescents by changing their living conditions. The present work investigates whether gender differences in selected health indicators are evident more than two years after the onset of the pandemic. Methods: In the study Kindergesundheit in Deutschland aktuell (KIDA) (German Children’s Health Update), cross-sectional telephone surveys were conducted with parents of 3- to 15-year-olds (n=3,478). Parental information on the general and mental health of the child, on increased need for health care and mental health services, as well as on physical activity and utilisation of sports activities were queried in standardised manner. Gender differences were assessed using Chi2 tests. Results: A total of 91% of the girls and 92% of the boys had their general health assessed as being (very) good by their parents (difference not significant, n.s.). An increased need for care and support was indicated for 10.6% of the 3- to 15-year-olds (girls: 9%, boys: 12%, n.s.). Boys met the physical activity recommendations of the WHO significantly more often (60%) than girls (54%). Good to excellent mental health was reported for 93% of both boys and girls. When changes during the pandemic were reported, no differences were found in the responses for girls compared to boys. Conclusions: Gender differences were found for individual parameters and age groups. These differences must be assessed in the context of other social determinants of health, and need to be considered when planning preventive measures

Culturally sensitive stepped care for adolescent refugees: efficacy and cost–utility of a multicentric randomized controlled trial

Adolescent refugees and asylum seekers (ARAS) are highly vulnerable to mental health problems. Stepped care models (SCM) and culturally sensitive therapies offer promising treatment approaches to effectively provide necessary medical and psychological support. To our knowledge, we were the first to investigate whether a culturally sensitive SCM will reduce symptoms of depression and PTSD in ARAS more effectively and efficiently than treatment as usual (TAU). We conducted a multicentric, randomized, controlled and rater-blinded trial across Germany with ARAS between the ages of 14 to 21 years. Participants (N = 158) were stratified by their level of depressive symptom severity and then equally randomized to either SCM or TAU. Depending on their severity level, SCM participants were allocated to tailored interventions. Symptom changes were assessed for depression (PHQ) and PTSD (CATS) at four time points, with the primary end point at post-intervention after 12 weeks. Based on an intention-to-treat sample, we used a linear mixed model approach for the main statistical analyses. Further evaluations included cost–utility analyses, sensitivity analyses, follow-up-analyses, response and remission rates and subgroup analysis. We found a significant reduction of PHQ (d = 0.52) and CATS (d = 0.27) scores in both groups. However, there was no significant difference between SCM and TAU. Cost–utility analyses indicated that SCM generated greater cost–utility when measured as quality-adjusted life years compared to TAU. Subgroup analysis revealed different effects for the SCM interventions depending on the outcome measure. Although culturally sensitive, SCMs did not prove to be more effective in symptom change and represent a more cost-effective treatment alternative for mentally burdened ARAS. Our research contributes to the optimization of clinical productivity and the improvement of therapeutic care for ARAS. Disorder-specific interventions should be further investigated