Into the unknown: expression profiling without genome sequence information in CHO by next generation sequencing.

The arrival of next-generation sequencing (NGS) technologies has led to novel opportunities for expression profiling and genome analysis by utilizing vast amounts of short read sequence data. Here, we demonstrate that expression profiling in organisms lacking any genome or transcriptome sequence information is feasible by combining Illumina's mRNA-seq technology with a novel bioinformatics pipeline that integrates assembled and annotated Chinese hamster ovary (CHO) sequences with information derived from related organisms. We applied this pipeline to the analysis of CHO cells which were chosen as a model system owing to its relevance in the production of therapeutic proteins. Specifically, we analysed CHO cells undergoing butyrate treatment which is known to affect cell cycle regulation and to increase the specific productivity of recombinant proteins. By this means, we identified sequences for >13,000 CHO genes which added sequence information of approximately 5000 novel genes to the CHO model. More than 6000 transcript sequences are predicted to be complete, as they covered >95% of the corresponding mouse orthologs. Detailed analysis of selected biological functions such as DNA replication and cell cycle control, demonstrated the potential of NGS expression profiling in organisms without extended genome sequence to improve both data quantity and quality.

CHO gene expression profiling in biopharmaceutical process analysis and design.

Increase in both productivity and product yields in biopharmaceutical process development with recombinant protein producing mammalian cells can be mainly attributed to the advancements in cell line development, media, and process optimization. Only recently, genome-scale technologies enable a system-level analysis to elucidate the complex biomolecular basis of protein production in mammalian cells promising an increased process understanding and the deduction of knowledge-based approaches for further process optimization. Here, the use of gene expression profiling for the analysis of a low titer (LT) and high titer (HT) fed batch process using the same IgG producing CHO cell line was investigated. We found that gene expression (i) significantly differed in HT versus LT process conditions due to differences in applied chemically defined, serum-free media, (ii) changed over the time course of the fed batch processes, and that (iii) both metabolic pathways and 14 biological functions such as cellular growth or cell death were affected. Furthermore, detailed analysis of metabolism in a standard process format revealed the potential use of transcriptomics for rational media design as is shown for the case of lipid metabolism where the product titer could be increased by about 20% based on a lipid modified basal medium. The results demonstrate that gene expression profiling can be an important tool for mammalian biopharmaceutical process analysis and optimization.

Actin/alpha-actinin-dependent transport of AMPA receptors in dendritic spines: role of the PDZ-LIM protein RIL.

The efficacy of excitatory transmission in the brain depends to a large extent on synaptic AMPA receptors, hence the importance of understanding the delivery and recycling of the receptors at the synaptic sites. Here we report a novel regulation of the AMPA receptor transport by a PDZ (postsynaptic density-95/Drosophila disc large tumor suppressor zona occludens 1) and LIM (Lin11/rat Isl-1/Mec3) domain-containing protein, RIL (reversion-induced LIM protein). We show that RIL binds to the AMPA glutamate receptor subunit GluR-A C-terminal peptide via its LIM domain and to alpha-actinin via its PDZ domain. RIL is enriched in the postsynaptic density fraction isolated from rat forebrain, strongly localizes to dendritic spines in cultured neurons, and coprecipitates, together with alpha-actinin, in a protein complex isolated by immunoprecipitation of AMPA receptors from forebrain synaptosomes. Functionally, in heterologous cells, RIL links AMPA receptors to the alpha-actinin/actin cytoskeleton, an effect that appears to apply selectively to the endosomal surface-internalized population of the receptors. In cultured neurons, an overexpression of recombinant RIL increases the accumulation of AMPA receptors in dendritic spines, both at the total level, as assessed by immunodetection of endogenous GluR-A-containing receptors, and at the synaptic surface, as assessed by recording of miniature EPSCs. Our results thus indicate that RIL directs the transport of GluR-A-containing AMPA receptors to and/or within dendritic spines, in an alpha-actinin/actin-dependent manner, and that such trafficking function promotes the synaptic accumulation of the receptors.

Advancing biopharmaceutical process development by system-level data analysis and integration of omics data.

Development of efficient bioprocesses is essential for cost-effective manufacturing of recombinant therapeutic proteins. To achieve further process improvement and process rationalization comprehensive data analysis of both process data and phenotypic cell-level data is essential. Here, we present a framework for advanced bioprocess data analysis consisting of multivariate data analysis (MVDA), metabolic flux analysis (MFA), and pathway analysis for mapping of large-scale gene expression data sets. This data analysis platform was applied in a process development project with an IgG-producing Chinese hamster ovary (CHO) cell line in which the maximal product titer could be increased from about 5 to 8 g/L.Principal component analysis (PCA), k-means clustering, and partial least-squares (PLS) models were applied to analyze the macroscopic bioprocess data. MFA and gene expression analysis revealed intracellular information on the characteristics of high-performance cell cultivations. By MVDA, for example, correlations between several essential amino acids and the product concentration were observed. Also, a grouping into rather cell specific productivity-driven and process control-driven processes could be unraveled. By MFA, phenotypic characteristics in glycolysis, glutaminolysis, pentose phosphate pathway, citrate cycle, coupling of amino acid metabolism to citrate cycle, and in the energy yield could be identified. By gene expression analysis 247 deregulated metabolic genes were identified which are involved, inter alia, in amino acid metabolism, transport, and protein synthesis.

Beneficial therapeutic effects with different particulate structures of murine polyomavirus VP1-coat protein carrying self or non-self CD8 T cell epitopes against murine melanoma.

Polyomavirus-like-particles (PLPs) are empty, non-replicative, non-infectious particles that represent a potent antigen-delivery system against malignant disease. Protective anti-tumour immunity can be induced under therapy conditions by subcutaneous (s.c.) treatment with particulate antigenic structures like chimerical polyomavirus-pentamers (PPs). These PPs displaying an immunodominant H-2Kb-restricted ovalbumin (OVA)257-264 epitope evoked nearly complete tumour remission in MO5 (B16-OVA) melanoma-bearing C57BL/6 mice by two s.c. applications in a weekly interval. The immunotherapeutic intervention started at day 4 after melanoma implant. Furthermore, 40% of melanoma-bearing mice vaccinated with heterologous PPs carrying a H-2Kb-restricted cytotoxic T lymphocyte (CTL) epitope derived from of tyrosinase-related protein 2 (TRP2) survived similar treatment conditions. However, a late immunotherapeutic onset at day 10 post melanoma inoculation revealed no significant differences between the therapeutic values (40-60% survival) of VP1-OVA252-270 and VP1-TRP2180-192 PPs, respectively. These experiments underlined the capacity of PPs to break T cell tolerance against a differentially expressed self-antigen. As a correlate for preventive and therapeutic immunity against MO5 melanoma the number of OVA257-264- or TRP2180-188-specific CD8 T cells were significantly increased within the splenocyte population of treated mice as measured by H-2Kb-OVA257-264-PE tetramer staining or appropriate ELISPOT assays, respectively. These results reveal that heterologous PLPs and even chimerical PPs represent highly efficient antigen carriers for inducing CTL responses underlining their potential as immunotherapeutics against cancer.