Enhancing rAAV production by HEK293 cells via metabolic profiling

Viral vector manufacturing is expensive and time-consuming. Demand for rAAV-based vectors has risen massively in the past decade and continues to rise thanks to urgent healthcare supply demand. The industry is, however, currently missing a cost-effective and robust manufacturing strategy. One of the major downsides of rAAV production is the high percentage of “empty” vector particles being produced and harvested. In addition to complicating downstream purification processes, this characteristic limits the efficiency of rAAV manufacture and presents uncertainties for scale-up. Efficiency of the manufacturing process is largely dependent on the productivity of the production cell line. Much emphasis has been put into understanding the effects of recombinant protein production on mammalian cell lines (e.g., CHO, HeLa, HEK293) but relatively little is known about the effects of viral vector production on cell biology and behaviour. Over the years, many clones have been derived, isolated and engineered from HEK293 to induce improvements in productivity and efficiency. However, the high cost of production and licensing, the expression of potentially undesired elements (e.g., T-antigen) and regulatory approval processes for next generation cell lines, hinders their use in clinical manufacturing. Increased understanding of HEK293 in relation to existing processes and process control offers realistic opportunity to enhance the efficiency of rAAV manufacturing. Our aim is to identify and understand the critical parameters that contribute to setting the productivity in HEK293 cells (in terms of final yield and abundance of full capsids), ranging from the metabolic requirements prior to and during viral vector production, to cell culture parameter optimisation to maintain the cells in an optimal state of health. We tested several commercially available media for rAAV9 production and selected the candidate that provided the best yield and quality of viral vector. With this medium as our baseline, we investigated the metabolism during a period of culture via extracellular metabolic profiling of control and rAAV producing cells. The analysis revealed the rapid use of several amino acids over the first 24 hr post-inoculation and the subsequent generation of metabolites indicative of metabolic profiles associated with cell growth. rAAV9 producing cells show lower rates of amino acid and glucose consumption than control cells but the profile of metabolism was not significantly changed as a result of transfection/production of rAAV9. These data were used to design medium supplements and the effect of supplement addition on cell proliferation, viability and rAAV production/quality was assessed. Specific combinations of amino acids generated an increased cell density (up to 9.3x106 cells/mL at 5 days post-inoculation compared to 4.4x106 cells/mL for cells in non-supplemented medium). This was associated with retention of improved viability in the presence of the supplement. In addition, the metabolic profiling we undertook indicated the build-up of potentially toxic/growth inhibitory metabolites during the period of stock cell preparations prior to setting up transfections. In various dilution experiments we were able to optimise the pre-treatment, cell density and dilution protocol to generate predictable and reproducible efficiencies of transfection, cell growth and rAAV production. Overall, our data contributes metabolic insights to process conditions that generate HEK293 cells of appropriate health and defined parameters to robust and enhanced production of rAAV, providing work schemes that are also appropriate to the manufacture of further types of viral vectors

Microcephaly with a disproportionate hippocampal reduction, stem cell loss and neuronal lipid droplet symptoms in Trappc9 KO mice

Mutations of the humanTRAFFICKING PROTEIN PARTICLE COMPLEX SUBUNIT 9(TRAPPC9) cause a neurodevelopmental disorder characterised by microcephaly and intellectual disability. Trappc9 constitutes a subunit specific to the intracellular membrane-associated TrappII complex. The TrappII complex interacts with Rab11 and Rab18, the latter being specifically associated with lipid droplets (LDs). Here we used non-invasive imaging to characteriseTrappc9knock-out (KO) mice as a model of the human hereditary disorder. KOs developed postnatal microcephaly with many grey and white matter regions being affected.In vivoMRI identified a disproportionately stronger volume reduction in the hippocampus, which was associated with a significant loss of Sox2-positive neural stem and progenitor cells. Diffusion Tensor imaging indicated a reduced organisation or integrity of white matter areas.Trappc9KOs displayed behavioural abnormalities in several tests related to exploration, learning and memory. Trappc9-deficient primary hippocampal neurons accumulated a larger LD volume per cell following Oleic Acid stimulation, and the coating of LDs by Perilipin-2 was much reduced. Additionally,Trappc9KOs developed obesity, which was significantly more severe in females than in males. Our findings indicate that, beyond previously reported Rab11-related vesicle transport defects, dysfunctions in LD homeostasis might contribute to the neurobiological symptoms of Trappc9 deficiency.</jats:p

Microcephaly with a disproportionate hippocampal reduction, stem cell loss and neuronal lipid droplet symptoms in Trappc9 KO mice

Mutations of the human TRAFFICKING PROTEIN PARTICLE COMPLEX SUBUNIT 9 (TRAPPC9) cause a neurodevelopmental disorder characterised by microcephaly and intellectual disability. Trappc9 constitutes a subunit specific to the intracellular membrane-associated TrappII complex. The TrappII complex interacts with Rab11 and Rab18, the latter being specifically associated with lipid droplets (LDs). Here we used non-invasive imaging to characterise Trappc9 knock-out (KO) mice as a model of the human hereditary disorder. KOs developed postnatal microcephaly with many grey and white matter regions being affected. In vivo magnetic resonance imaging (MRI) identified a disproportionately stronger volume reduction in the hippocampus, which was associated with a significant loss of Sox2-positive neural stem and progenitor cells. Diffusion tensor imaging indicated a reduced organisation or integrity of white matter areas. Trappc9 KOs displayed behavioural abnormalities in several tests related to exploration, learning and memory. Trappc9-deficient primary hippocampal neurons accumulated a larger LD volume per cell following Oleic Acid stimulation, and the coating of LDs by Perilipin-2 was much reduced. Additionally, Trappc9 KOs developed obesity, which was significantly more severe in females than in males. Our findings indicate that, beyond previously reported Rab11-related vesicle transport defects, dysfunctions in LD homeostasis might contribute to the neurobiological symptoms of Trappc9 deficiency

Reductions in hypothalamic Gfap expression, glial cells and α-tanycytes in lean and hypermetabolic Gnasxl-deficient mice

BACKGROUND: Neuronal and glial differentiation in the murine hypothalamus is not complete at birth, but continues over the first two weeks postnatally. Nutritional status and Leptin deficiency can influence the maturation of neuronal projections and glial patterns, and hypothalamic gliosis occurs in mouse models of obesity. Gnasxl constitutes an alternative transcript of the genomically imprinted Gnas locus and encodes a variant of the signalling protein Gαs, termed XLαs, which is expressed in defined areas of the hypothalamus. Gnasxl-deficient mice show postnatal growth retardation and undernutrition, while surviving adults remain lean and hypermetabolic with increased sympathetic nervous system (SNS) activity. Effects of this knock-out on the hypothalamic neural network have not yet been investigated. RESULTS: RNAseq analysis for gene expression changes in hypothalami of Gnasxl-deficient mice indicated Glial fibrillary acid protein (Gfap) expression to be significantly down-regulated in adult samples. Histological analysis confirmed a reduction in Gfap-positive glial cell numbers specifically in the hypothalamus. This reduction was observed in adult tissue samples, whereas no difference was found in hypothalami of postnatal stages, indicating an adaptation in adult Gnasxl-deficient mice to their earlier growth phenotype and hypermetabolism. Especially noticeable was a loss of many Gfap-positive α-tanycytes and their processes, which form part of the ependymal layer that lines the medial and dorsal regions of the 3(rd) ventricle, while β-tanycytes along the median eminence (ME) and infundibular recesses appeared unaffected. This was accompanied by local reductions in Vimentin and Nestin expression. Hypothalamic RNA levels of glial solute transporters were unchanged, indicating a potential compensatory up-regulation in the remaining astrocytes and tanycytes. CONCLUSION: Gnasxl deficiency does not directly affect glial development in the hypothalamus, since it is expressed in neurons, and Gfap-positive astrocytes and tanycytes appear normal during early postnatal stages. The loss of Gfap-expressing cells in adult hypothalami appears to be a consequence of the postnatal undernutrition, hypoglycaemia and continued hypermetabolism and leanness of Gnasxl-deficient mice, which contrasts with gliosis observed in obese mouse models. Since α-tanycytes also function as adult neural progenitor cells, these findings might indicate further developmental abnormalities in hypothalamic formations of Gnasxl-deficient mice, potentially including neuronal composition and projections

Molecular characterization of HEK293 cells as emerging versatile cell factories.

From PubMed via Jisc Publications RouterHistory: received 2021-03-05, revised 2021-04-29, accepted 2021-05-07Publication status: aheadofprintHEK293 cell lines are used for the production of recombinant proteins, virus-like particles and viral vectors. Recent work has generated molecular (systems level) characterisation of HEK293 variants that has enabled re-engineering of the cells towards enhanced use for manufacture-scale production of recombinant biopharmaceuticals (assessment of 'safe harbours' for gene insertion, engineering of new variants for stable, amplifiable expression). In parallel, there have been notable advances in the bioprocessing conditions (suspension adaptation, development of defined serum-free media) that offer the potential for large-scale manufacture, a feature especially important in the drive to produce viral vectors at large-scale and at commercially viable costs for gene therapy. The combination of cell-based and bioprocess-based modification of existing HEK293 cell processes, frequently informed by understandings transferred from developments with Chinese hamster ovary cell lines, seems destined to place the HEK293 cell systems firmly as a critical platform for production of future biologically based therapeutics. [Abstract copyright: Copyright © 2021 Elsevier Ltd. All rights reserved.

Variable allelic expression of imprinted genes at the Peg13, Trappc9, Ago2 cluster in single neural cells.

Peer reviewed: TrueAcknowledgements: We would like to thank all staff of the animal facility for their dedicated work. We would also like to thank Abigail Clark and Megan Green for contributions to early stages of the project.Genomic imprinting is an epigenetic process through which genes are expressed in a parent-of-origin specific manner resulting in mono-allelic or strongly biased expression of one allele. For some genes, imprinted expression may be tissue-specific and reliant on CTCF-influenced enhancer-promoter interactions. The Peg13 imprinting cluster is associated with neurodevelopmental disorders and comprises canonical imprinted genes, which are conserved between mouse and human, as well as brain-specific imprinted genes in mouse. The latter consist of Trappc9, Chrac1 and Ago2, which have a maternal allelic expression bias of ∼75% in brain. Findings of such allelic expression biases on the tissue level raise the question of how they are reflected in individual cells and whether there is variability and mosaicism in allelic expression between individual cells of the tissue. Here we show that Trappc9 and Ago2 are not imprinted in hippocampus-derived neural stem cells (neurospheres), while Peg13 retains its strong bias of paternal allele expression. Upon analysis of single neural stem cells and in vitro differentiated neurons, we find not uniform, but variable states of allelic expression, especially for Trappc9 and Ago2. These ranged from mono-allelic paternal to equal bi-allelic to mono-allelic maternal, including biased bi-allelic transcriptional states. Even Peg13 expression deviated from its expected paternal allele bias in a small number of cells. Although the cell populations consisted of a mosaic of cells with different allelic expression states, as a whole they reflected bulk tissue data. Furthermore, in an attempt to identify potential brain-specific regulatory elements across the Trappc9 locus, we demonstrate tissue-specific and general silencer activities, which might contribute to the regulation of its imprinted expression bias

Additional file 4: of Reductions in hypothalamic Gfap expression, glial cells and α-tanycytes in lean and hypermetabolic Gnasxl-deficient mice

Gnasxl is not expressed in the ependymal layer, but the parenchyma of the hypothalamus. IHC (A) for XLαs (green) in the postnatal day 1 (P1) hypothalamus and in situ hybridisation for Gnasxl at P4 (B) show scattered positive cells in the DMH, but not in the ependymal layer (ep) of the 3rd ventricle (DAPI nuclear counterstain). (C) Similarly, IHC for XLαs in the adult hypothalamus marks neurons in the parenchyma, but no expression is found in ependymal cells (arrow). XLαs is a membrane-associated protein detectable in neurites, but no tanycyte extensions are stained. Arc = arcuate nucleus, DMH = dorsomedial nucleus, ME = median eminence. Scale bar = 100 μm. (PDF 619 kb