Optimization of rAAV mediated targeted suicide gene therapy, rAAV manufacturing and downstream processing

Teschner K. Optimization of rAAV mediated targeted suicide gene therapy, rAAV manufacturing and downstream processing. Bielefeld: Universität Bielefeld; 2020.Recombinant adeno-associated viruses (rAAV) have gained an outstanding reputation in gene therapy due to their low immunogenicity, high stability and long-term gene expression in target cells. However, there is a lack of efficient and cost-effective manufacturing processes to enable the expansion of AAVs into therapeutic indications such as tumor therapy where higher doses are needed. Additionally, in tumor therapy, the broad tropism of the AAV is problematic since harmful effects on healthy cells can be expected during cancer gene therapy with lethal transgenes. In this work, the use of tumor-specific promoters (TSPs) and microRNA (miRNA) target sequences have been analyzed to allow selective eradication of tumor cells. Moreover, the use of a rAAV production cell line generated in our laboratory, a new affinity chromatography and the possibility of rAAV production in suspension cells have been analyzed. For efficient rAAV production, a new cell line with integrated adenoviral helper sequences was analyzed during this work. Providing a RepCap and an ITR/vector plasmid during transient transfection was established, leading to a significant reduction in the amount of plasmid DNA required for production. Biological characteristics and the yield were analyzed and were comparable to those obtained with the triple transfection protocol in the HEK-293 standard cell line. This result demonstrates that the new producer ensures constant quality of viral particles produced while reducing costs and time, as providing of the helper plasmid is no longer required. Upscaling of the production is very restricted due to the limited growth area in the standard adherent growing HEK-293 cells. Therefore, a triple transfection protocol for the rAAV production was established, in a commercially available suspension adapted cell line and the medium conditions for cell propagation and production were adjusted and resulted in a protocol with increased yields compared to the starting conditions. The high cell density cultivation in the suspension-based process led to a higher volumetric yield than the process based on adherent growing cells and at the same time allows for up-scaling. The use of single domains of the natural AAV receptor (AAVR) as an affinity ligand for rAAV purification was established in this work. Different carrier materials for covalent binding were tested, with cellulose paper showing the best characteristics regarding the ease of handling as well as final purity and yield of the rAAV sample. The biological activity of rAAV purified by this affinity chromatography was comparable to those purified by the standard ultracentrifugation (UC) method but with a superior purity. Furthermore, the processing time was reduced from more than one day for UC purification to a few hours for the affinity chromatography, since the affinity chromatography was optimized for the application of directly capturing rAAVs from the crude cell extract contrary to UC purification where a concentrating step is necessary. The benefits of TSPs and miRNA mediated de-targeting in a virus-directed enzyme prodrug therapy (VDEPT) were investigated in this work. As a suicide gene, the herpes simplex virus thymidinekinase (HSV-tk) was used, and the conversion of the prodrug Ganciclovir (GCV) to its toxic metabolite was indirectly measured by a cytotoxicity assay. The activity of three tumor-specific promoters (survivin (SUR), cyclooxygenase-2 (COX-2) and C-X-C motif chemokine receptor 4 (CXCR-4)) was compared to the cytomegalovirus immediate early promoter (CMV) in four cancer and two healthy cell lines. For SUR and CMV promoter driven HSV-tk expression, a let-7a miRNA target sequence was included. Depending on the cell line investigated, tumor specificity was enabled, with the CMV promoter driven gene expression in combination with the let-7a miRNA target sequence reaching, the highest tumor specificity in all investigated cancer cell lines. The results of this study provide a starting point for further improvements of the rAAV manufacturing process and strategies for de-targeting of healthy cells in VDEPT approaches. For example, an adaption of the new rAAV production cell line to suspension and combinations of CMV promoter driven gene expression with distinct miRNA target sequences could be tested. In addition, combinations of transcriptional targeting and miRNA mediated de-targeting should be tested together with tumor marker-based re-targeting of rAAVs, which may enable more specific suicide gene therapy in the future

Cohort Fertility Patterns in the Nordic Countries

Previous analyses of period fertility suggest that the trends of the Nordic countries are sufficiently similar that we may speak of a common "Nordic fertility regime". We investigate whether this assumption can be corroborated by comparing cohort fertility patterns in the Nordic countries. We study cumulated and completed fertility of Nordic birth cohorts based on the childbearing histories of women born in 1935 and later derived from the population registers of Denmark, Finland, Norway, and Sweden. We further explore childbearing behaviour by women’s educational attainment. The results show remarkable similarities in postponement and recuperation between the countries. Median childbearing age is about two to three years higher in the 1960−64 cohort than in the 1950−54 cohort, but the younger cohort recuperates the fertility level of the older cohort at ages 30 and above. A similar pattern of recuperation can be observed for highly educated women compared to women with less education, resulting in small differences in completed fertility across educational groups. Another interesting finding is that of a positive relationship between educational level and the final number of children when women who become mothers at similar ages are compared. Despite some differences in the levels of childlessness, country differences in fertility outcome are generally small. The cohort analyses thus support the notion of a common Nordic fertility regime.cohort fertility, educational attainment, Nordic countries, postponement, recuperation

Cohort fertility patterns in the Nordic Countries

Previous analyses of period fertility suggest that the trends of the Nordic countries are sufficiently similar to speak of a common "Nordic fertility regime". We investigate whether this assumption can be corroborated by comparing cohort fertility patterns in the Nordic countries. We study cumulated and completed fertility of Nordic birth cohorts based on the childbearing histories of women born in 1935 and later derived from the population registers of Denmark, Finland, Norway, and Sweden. We further explore childbearing behaviour by womenâs educational attainment. The results show remarkable similarities in postponement and recuperation between the countries and very small differences in completed fertility across educational groups. Median childbearing age is about 2−3 years higher in the 1960−64 cohort than in the 1950−54 cohort, but the younger cohort recuperates the fertility level of the older cohort at ages 30 and above. A similar pattern of recuperation can be observed for highly educated women as compared to women with less education. An interesting finding is that of a positive relationship between educational level and the final number of children when women who become mothers at similar ages are compared. Country differences in fertility outcome are generally rather low. Childlessness is highest in Finland and lowest in Norway, and the educational differentials are largest in Norway. Despite such differences, the cohort analyses in many ways support the notion of a common Nordic fertility regime.Denmark, Finland, Norway, Sweden, cohort fertility

On the AAVenue to success: Advances in technologies for AAV production

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rAAV Engineering for Capsid-Protein Enzyme Insertions and Mosaicism Reveals Resilience to Mutational, Structural and Thermal Perturbations

Feiner R, Teschner J, Teschner K, et al. rAAV Engineering for Capsid-Protein Enzyme Insertions and Mosaicism Reveals Resilience to Mutational, Structural and Thermal Perturbations. International Journal of Molecular Sciences. 2019;20(22): 5702.Recombinant adeno-associated viruses (rAAV) provide outstanding options for customization and superior capabilities for gene therapy. To access their full potential, facile genetic manipulation is pivotal, including capsid loop modifications. Therefore, we assessed capsid tolerance to modifications of the structural VP proteins in terms of stability and plasticity. Flexible glycine-serine linkers of increasing sizes were, at the genetic level, introduced into the 587 loop region of the VP proteins of serotype 2, the best studied AAV representative. Analyses of biological function and thermal stability with respect to genome release of viral particles revealed structural plasticity. In addition, insertion of the 29 kDa enzyme β-lactamase into the loop region was tested with a complete or a mosaic modification setting. For the mosaic approach, investigation of VP2 trans expression revealed that a Kozak sequence was required to prevent leaky scanning. Surprisingly, even the full capsid modification with β-lactamase allowed for the assembly of capsids with a concomitant increase in size. Enzyme activity assays revealed lactamase functionality for both rAAV variants, which demonstrates the structural robustness of this platform technology.</jats:p

rAAV for Tumor Therapy Using Transcriptional and Translational Control of Suicide Gene Expression Purified by a Newly Developed Affinity Chromatography Based on the PKD Domains of AAVR

Teschner K, Teschner J, Leppin M, Müller K. rAAV for Tumor Therapy Using Transcriptional and Translational Control of Suicide Gene Expression Purified by a Newly Developed Affinity Chromatography Based on the PKD Domains of AAVR. In: MOLECULAR THERAPY. Vol 28. Cambridge: Cell Press; 2020: 423

Synthesis and Concomitant Assembly of Adeno-Associated Virus-like Particles in Escherichia coli

Le DT, Radukic M, Teschner K, Becker L, Müller K. Synthesis and Concomitant Assembly of Adeno-Associated Virus-like Particles in Escherichia coli . ACS Synthetic Biology . 2022.Virus-like particles (VLPs) have been used for numerous pharmaceutical applications, particularly vaccination and drug delivery. Recombinant adeno-associated virus (rAAV), a leading candidate in gene therapy, has been proposed as a vaccine scaffold, but high production costs limit its use. Here we establish intracellular production of AAV VLPs in Escherichia coli. VP3 capsid proteins of AAV serotype 5 (AAV5) were expressed, and VLPs were readily purified. The correct assembly was confirmed by ELISA with an intact-capsid AAV5 antibody and an AAVR domain as well as by atomic force microscopy. Biological functionality was demonstrated with a HeLa cell internalization assay. Coexpression of the assembly-activating protein (AAP) of AAV5 in E. coli improved capsid yield. This work provides the first evidence that AAV VLPs form in E. coli, opening new opportunities for production and exploration of AAV VLPs for biomedical applications

Cohort Fertility Patterns in the Nordic Countries

Previous analyses of period fertility suggest that the trends of the Nordic countries are sufficiently similar to speak of a common &quot;Nordic fertility regime&quot;. We investigate whether this assumption can be corroborated by comparing cohort fertility patterns in the Nordic countries. We study cumulated and completed fertility of Nordic birth cohorts based on the childbearing histories of women born in 1935 and later derived from the population registers of Denmark, Finland, Norway, and Sweden. We further explore childbearing behaviour by women&apos;s educational attainment. The results show remarkable similarities in postponement and recuperation between the countries and very small differences in completed fertility across educational groups. Median childbearing age is about 2-3 years higher in the 1960-64 cohort than in the 1950-54 cohort, but the younger cohort recuperates the fertility level of the older cohort at ages 30 and above. A similar pattern of recuperation can be observed for highly educated women as compared to women with less education. An interesting finding is that of a positive relationship between educational level and the final number of children when women who become mothers at similar ages are compared. Country differences in fertility outcome are generally rather low. Childlessness is highest in Finland and lowest in Norway, and the educational differentials are largest in Norway. Despite such differences, the cohort analyses in many ways support the notion of a common Nordic fertility regime

Indicators of acute and persistent renal damage in adult thrombotic microangiopathy.

BACKGROUND: Thrombotic microangiopathies (TMA) in adults such as thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS) are life-threatening disorders if untreated. Clinical presentation is highly variable and prognostic factors for clinical course and outcome are not well established. METHODS: We performed a retrospective observational study of 62 patients with TMA, 22 males and 40 females aged 16 to 76 years, treated with plasma exchange at one center to identify clinical risk factors for the development of renal insufficiency. RESULTS: On admission, 39 of 62 patients (63%) had acute renal failure (ARF) with 32 patients (52%) requiring dialysis treatment. High systolic arterial pressure (SAP, p = 0.009) or mean arterial pressure (MAP, p = 0.027) on admission was associated with acute renal failure. Patients with SAP>140 mmHg on admission had a sevenfold increased risk of severe kidney disease (OR 7.464, CI 2.097-26.565). MAP>100 mmHg indicated a fourfold increased risk for acute renal failure (OR 4.261, CI 1.400-12.972). High SAP, diastolic arterial pressure (DAP), and MAP on admission were also independent risk factors for persistent renal insufficiency with the strongest correlation for high MAP. Moreover, a high C-reactive protein (CRP) level on admission correlated with renal failure in the course of the disease (p = 0.003). At discharge, renal function in 11 of 39 patients (28%) had fully recovered, 14 patients (23%) remained on dialysis, and 14 patients (23%) had non-dialysis-dependent chronic kidney disease. Seven patients (11%) died. We identified an older age as risk factor for death. CONCLUSIONS: High blood pressure as well as high CRP serum levels on admission are associated with renal insufficiency in TMA. High blood pressure on admission is also a strong predictor of sustained renal insufficiency. Thus, adult TMA patients with high blood pressure may require special attention to prevent persistent renal failure

Characteristic factors for acute renal failure on admission.

<p>*values significantly higher in patients with impaired renal function compared to patients with normal renal function on admission (p<0.05). IQR: Interquartile range.</p