Development of Novel Transient Delivery Systems for Gene Therapy

Gene and cell therapies hold enormous promise for many presently incurable diseases. Although the efficiency and safety of site-specific genome editing systems have improved tremendously over the past decade, the delivery of these novel technologies to specific target cells remains a key technical bottleneck for the clinical translation. Since long-term expression of DNA-modifying enzymes can be associated with cytotoxicity, transient delivery is desirable to reduce the risk of mutagenesis from off-target activity. A main target for gene and cell therapies are T cells. In order to achieve gene transfer into human T cells, current gene delivery techniques involve significant levels of T cell activation and ex vivo expansion prior to lentiviral or retroviral transduction. However, ex vivo stimulation and expansion have been shown to significantly alter the natural physiology of T cells. To address these challenges, we have developed a non-integrative lentiviral gene therapy platform to focus on genetic modifications of resting CD4+ T cells. To overcome barriers, present in this notoriously hard to transduce cell population, we have re-engineered lentiviral particles from the inside-out. Firstly, we have identified key viral glycoproteins that can mediate cytosolic delivery of transgenes across resting CD4+ T cell plasma membranes. Secondly, by replacing HIV nucleocapsid with the coat protein of the MS2 bacteriophage and substituting lentiviral RNA packaging motifs (LTR and packaging signal Ψ) with a series of MS2 stem loops that facilitate RNA binding to the MS2 coat protein, we have enabled the packaging of CRISPR/Cas9 components into these particles. In this setting, we observed expression of biologically active mRNA in up to 65% of resting CD4+ T cells. Furthermore, with our CRISPR/Cas9 delivery platform we have achieved genome editing efficiencies of >85% in human cell lines, and >20% in primary CD4+ T cells. With the emergence of SARS-CoV-2, we have shifted our focus on the development of a novel viral vector for gene delivery. Using a SARS-CoV-2 VLP scaffold, we have enabled the packaging and transient delivery of mRNA into target cells by tethering a reporter gene to the recently identified SARS-CoV-2 packaging signal. Equipping these VLPs with Spike glycoproteins from emerging SARS-CoV-2 variants, we observed an increase in transient mRNA delivery to >50% of target cells. This platform may give us a novel set of viral vector solutions to use for many respiratory diseases, including coronavirus disease (COVID-19) itself. Taken together, this thesis describes the development of robust delivery platforms for the transient delivery of biologically active transgenes to multiple cell types

Operationalization of a multidimensional sex/gender concept for quantitative environmental health research and implementation in the KORA study: Results of the collaborative research project INGER

BackgroundIn environmental health research, sex and gender are not yet adequately considered. There is a need to improve data collection in population-based environmental health studies by comprehensively surveying sex/gender-related aspects according to gender theoretical concepts. Thus, within the joint project INGER we developed a multidimensional sex/gender concept which we aimed to operationalize and to test the operationalization for feasibility.MethodsIn an iterative process, we created questionnaire modules which quantitatively captured the requirements of the INGER sex/gender concept. We deployed it in the KORA cohort (Cooperative Health Research in the Region of Augsburg, Germany) in 2019 and evaluated response and missing rates.ResultsThe individual sex/gender self-concept was surveyed via a two-step approach that asked for sex assigned at birth and the current sex/gender identity. Additionally, we used existing tools to query internalized sex/gender roles and externalized sex/gender expressions. Adapted to the KORA population, we asked for discrimination experiences and care and household activities contributing to explain structural sex/gender relations. Further intersectionality-related social categories (e.g., socio-economic position), lifestyle and psychosocial factors were covered through data available in KORA. We could not identify appropriate tools to assess the true biological sex, sexual orientation and ethnic/cultural identity, which have yet to be developed or improved. The response-rate was 71%, the evaluation of 3,743 questionnaires showed a low missing rate. Prevalence of marginalized groups regarding sex/gender identity and definable by experiences of discrimination was very low.ConclusionWe have shown how the multidimensional INGER sex/gender concept can be operationalized according to an European and North American understanding of sex/gender for use in quantitative research. The questionnaire modules proved feasible in an epidemiologic cohort study. Being a balancing act between theoretical concepts and its quantitative implementation our operationalization paves the way for an adequate consideration of sex/gender in environmental health research

SARS-CoV-2 neutralizing antibodies : longevity, breadth, and evasion by emerging viral variants

The Severe Acute Respiratory Syndrome Coronavirus 2 (SAU ARS-CoV-2) antibody neutralization response and its evasion by emerging viral variants and variant of concern (VOC) are unknown, but critical to understand reinfection risk and breakthrough infection following vaccination. Antibody immunoreactivity against SARS-CoV-2 antigens and Spike variants, inhibition of Spike-driven virus–cell fusion, and infectious SARS-CoV-2 neutralization were characterized in 807 serial samples from 233 reverse transcription polymerase chain reaction (RT-PCR)–confirmed Coronavirus Disease 2019 (COVID-19) individuals with detailed demographics and followed up to 7 months. A broad and sustained polyantigenic immunoreactivity against SARS-CoV-2 Spike, Membrane, and Nucleocapsid proteins, along with high viral neutralization, was associated with COVID-19 severity. A subgroup of “high responders” maintained high neutralizing responses over time, representing ideal convalescent plasma donors. Antibodies generated against SARS-CoV-2 during the first COVID-19 wave had reduced immunoreactivity and neutralization potency to emerging Spike variants and VOC. Accurate monitoring of SARS-CoV-2 antibody responses would be essential for selection of optimal responders and vaccine monitoring and design

Genomic Relationships, Novel Loci, and Pleiotropic Mechanisms across Eight Psychiatric Disorders

Genetic influences on psychiatric disorders transcend diagnostic boundaries, suggesting substantial pleiotropy of contributing loci. However, the nature and mechanisms of these pleiotropic effects remain unclear. We performed analyses of 232,964 cases and 494,162 controls from genome-wide studies of anorexia nervosa, attention-deficit/hyper-activity disorder, autism spectrum disorder, bipolar disorder, major depression, obsessive-compulsive disorder, schizophrenia, and Tourette syndrome. Genetic correlation analyses revealed a meaningful structure within the eight disorders, identifying three groups of inter-related disorders. Meta-analysis across these eight disorders detected 109 loci associated with at least two psychiatric disorders, including 23 loci with pleiotropic effects on four or more disorders and 11 loci with antagonistic effects on multiple disorders. The pleiotropic loci are located within genes that show heightened expression in the brain throughout the lifespan, beginning prenatally in the second trimester, and play prominent roles in neurodevelopmental processes. These findings have important implications for psychiatric nosology, drug development, and risk prediction.Peer reviewe

Dissecting the Shared Genetic Architecture of Suicide Attempt, Psychiatric Disorders, and Known Risk Factors

Background Suicide is a leading cause of death worldwide, and nonfatal suicide attempts, which occur far more frequently, are a major source of disability and social and economic burden. Both have substantial genetic etiology, which is partially shared and partially distinct from that of related psychiatric disorders. Methods We conducted a genome-wide association study (GWAS) of 29,782 suicide attempt (SA) cases and 519,961 controls in the International Suicide Genetics Consortium (ISGC). The GWAS of SA was conditioned on psychiatric disorders using GWAS summary statistics via multitrait-based conditional and joint analysis, to remove genetic effects on SA mediated by psychiatric disorders. We investigated the shared and divergent genetic architectures of SA, psychiatric disorders, and other known risk factors. Results Two loci reached genome-wide significance for SA: the major histocompatibility complex and an intergenic locus on chromosome 7, the latter of which remained associated with SA after conditioning on psychiatric disorders and replicated in an independent cohort from the Million Veteran Program. This locus has been implicated in risk-taking behavior, smoking, and insomnia. SA showed strong genetic correlation with psychiatric disorders, particularly major depression, and also with smoking, pain, risk-taking behavior, sleep disturbances, lower educational attainment, reproductive traits, lower socioeconomic status, and poorer general health. After conditioning on psychiatric disorders, the genetic correlations between SA and psychiatric disorders decreased, whereas those with nonpsychiatric traits remained largely unchanged. Conclusions Our results identify a risk locus that contributes more strongly to SA than other phenotypes and suggest a shared underlying biology between SA and known risk factors that is not mediated by psychiatric disorders.Peer reviewe

Rapid spread of the SARS-CoV-2 JN.1 lineage is associated with increased neutralization evasion

Summary: In July/August 2023, the highly mutated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) BA.2.86 lineage emerged and its descendant JN.1 is on track to become the dominant SARS-CoV-2 lineage globally. Compared to the spike (S) protein of the parental BA.2.86 lineage, the JN.1 S protein contains one mutation, L455S, which may affect receptor binding and antibody evasion. Here, we performed a virological assessment of the JN.1 lineage employing pseudovirus particles bearing diverse SARS-CoV-2 S proteins. Using this strategy, it was found that S protein mutation L455S confers increased neutralization resistance but reduces ACE2 binding capacity and S protein-driven cell entry efficiency. Altogether, these data suggest that the benefit of increased antibody evasion outweighs the reduced ACE2 binding capacity and further enabled the JN.1 lineage to effectively spread in the human population

Data_Sheet_1_Operationalization of a multidimensional sex/gender concept for quantitative environmental health research and implementation in the KORA study: Results of the collaborative research project INGER.pdf

BackgroundIn environmental health research, sex and gender are not yet adequately considered. There is a need to improve data collection in population-based environmental health studies by comprehensively surveying sex/gender-related aspects according to gender theoretical concepts. Thus, within the joint project INGER we developed a multidimensional sex/gender concept which we aimed to operationalize and to test the operationalization for feasibility.MethodsIn an iterative process, we created questionnaire modules which quantitatively captured the requirements of the INGER sex/gender concept. We deployed it in the KORA cohort (Cooperative Health Research in the Region of Augsburg, Germany) in 2019 and evaluated response and missing rates.ResultsThe individual sex/gender self-concept was surveyed via a two-step approach that asked for sex assigned at birth and the current sex/gender identity. Additionally, we used existing tools to query internalized sex/gender roles and externalized sex/gender expressions. Adapted to the KORA population, we asked for discrimination experiences and care and household activities contributing to explain structural sex/gender relations. Further intersectionality-related social categories (e.g., socio-economic position), lifestyle and psychosocial factors were covered through data available in KORA. We could not identify appropriate tools to assess the true biological sex, sexual orientation and ethnic/cultural identity, which have yet to be developed or improved. The response-rate was 71%, the evaluation of 3,743 questionnaires showed a low missing rate. Prevalence of marginalized groups regarding sex/gender identity and definable by experiences of discrimination was very low.ConclusionWe have shown how the multidimensional INGER sex/gender concept can be operationalized according to an European and North American understanding of sex/gender for use in quantitative research. The questionnaire modules proved feasible in an epidemiologic cohort study. Being a balancing act between theoretical concepts and its quantitative implementation our operationalization paves the way for an adequate consideration of sex/gender in environmental health research.</p

Predictive score for the development or progression of Graves' orbitopathy in patients with newly diagnosed Graves' hyperthyroidism

OBJECTIVE : To construct a predictive score for the development or progression of Graves’ orbitopathy (GO) in Graves’ hyperthyroidism (GH). DESIGN : Prospective observational study in patients with newly diagnosed GH, treated with antithyroid drugs (ATD) for 18 months at ten participating centers from EUGOGO in 8 European countries. METHODS : 348 patients were included with untreated GH but without obvious GO. Mixed effects logistic regression was used to determine the best predictors. A predictive score (called PREDIGO) was constructed. RESULTS : GO occurred in 15% (mild in 13% and moderate to severe in 2%), predominantly at 6–12 months after start of ATD. Independent baseline determinants for the development of GO were clinical activity score (assigned 5 points if score > 0), TSH-binding inhibitory immunoglobulins (2 points if TBII 2–10 U/L, 5 points if TBII > 10 U/L), duration of hyperthyroid symptoms (1 point if 1–4 months, 3 points if >4 months) and smoking (2 points if current smoker). Based on the odds ratio of each of these four determinants, a quantitative predictive score (called PREDIGO) was constructed ranging from 0 to 15 with higher scores denoting higher risk; positive and negative predictive values were 0.28 (95% CI 0.20–0.37) and 0.91 (95% CI 0.87–0.94) respectively. CONCLUSIONS : In patients without GO at diagnosis, 15% will develop GO (13% mild, 2% moderate to severe) during subsequent treatment with ATD for 18 months. A predictive score called PREDIGO composed of four baseline determinants was better in predicting those patients who will not develop obvious GO than who will

Predictive score for the development or progression of Graves' orbitopathy in patients with newly diagnosed Graves' hyperthyroidism

Objective: To construct a predictive score for the development or progression of Graves' orbitopathy (GO) in Graves' hyperthyroidism (GH). Design: Prospective observational study in patients with newly diagnosed GH, treated with antithyroid drugs (ATD) for 18 months at ten participating centers from EUGOGO in 8 European countries. Methods: 348 patients were included with untreated GH but without obvious GO. Mixed effects logistic regression was used to determine the best predictors. A predictive score (called PREDIGO) was constructed. Results: GO occurred in 15\% (mild in 13\% and moderate to severe in 2\%), predominantly at 6-12 months after start of ATD. Independent baseline determinants for the development of GO were clinical activity score (assigned 5 points if score> 0), TSH-binding inhibitory immunoglobulins (2 points if TBII 2-10 U/L, 5 points if TBII >10 U/L), duration of hyperthyroid symptoms (1 point if 1-4 months, 3 points if >4 months) and smoking (2 points if current smoker). Based on the odds ratio of each of these four determinants, a quantitative predictive score (called PREDIGO) was constructed ranging from 0 to 15 with higher scores denoting higher risk; positive and negative predictive values were 0.28 (95\% CI 0.20-0.37) and 0.91 (95\% CI 0.87-0.94) respectively. Conclusions: In patients without GO at diagnosis, 15\% will develop GO (13\% mild, 2\% moderate to severe) during subsequent treatment with ATD for 18 months. A predictive score called PREDIGO composed of four baseline determinants was better in predicting those patients who will not develop obvious GO than who will