Production of retroviral vectors in continuous high cell density culture.

Retroviral vectors derived from murine leukemia virus (MLV) are used in somatic gene therapy applications e.g. for genetic modification of hematopoietic stem cells. Recently, we reported on the establishment of a suspension viral packaging cell line (VPC) for the production of MLV vectors. Human embryonic kidney 293-F (HEK293-F) cells were genetically modified for this purpose using transposon vector technology. Here, we demonstrate the establishment of a continuous high cell density (HCD) process using this cell line. First, we compared different media regarding the maximum achievable viable cell concentration (VCC) in small scale. Next, we transferred this process to a stirred tank bioreactor before we applied intensification strategies. Specifically, we established a perfusion process using an alternating tangential flow filtration system. Here, VCCs up to 27.4E + 06 cells/mL and MLV vector titers up to 8.6E + 06 transducing units/mL were achieved. Finally, we established a continuous HCD process using a tubular membrane for cell retention and continuous viral vector harvesting. Here, the space-time yield was 18-fold higher compared to the respective batch cultivations. Overall, our results clearly demonstrate the feasibility of HCD cultivations for high yield production of viral vectors, especially when combined with continuous viral vector harvesting. KEY POINTS: • A continuous high cell density process for MLV vector production was established • The tubular cell retention membrane allowed for continuous vector harvesting • The established process had a 18-fold higher space time yield compared to a batch.

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.

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.

Infectious RNA: Human Immunodeficiency Virus (HIV) Biology, Therapeutic Intervention, and the Quest for a Vaccine.

Different mechanisms mediate the toxicity of RNA. Genomic retroviral mRNA hijacks infected host cell factors to enable virus replication. The viral genomic RNA of the human immunodeficiency virus (HIV) encompasses nine genes encoding in less than 10 kb all proteins needed for replication in susceptible host cells. To do so, the genomic RNA undergoes complex alternative splicing to facilitate the synthesis of the structural, accessory, and regulatory proteins. However, HIV strongly relies on the host cell machinery recruiting cellular factors to complete its replication cycle. Antiretroviral therapy (ART) targets different steps in the cycle, preventing disease progression to the acquired immunodeficiency syndrome (AIDS). The comprehension of the host immune system interaction with the virus has fostered the development of a variety of vaccine platforms. Despite encouraging provisional results in vaccine trials, no effective vaccine has been developed, yet. However, novel promising vaccine platforms are currently under investigation.

Establishment of a novel stable human suspension packaging cell line producing ecotropic retroviral MLV(PVC-211) vectors efficiently transducing murine hematopoietic stem and progenitor cells.

Retroviral vectors derived from murine leukemia virus (MLV) are amongst the most frequently utilized vectors in gene therapy approaches such as the genetic modification of hematopoietic cells. Currently, vector particles are mostly produced employing adherent viral packaging cell lines (VPCs) rendering the scale up of production laborious, and thus cost-intensive. Here, we describe the rapid establishment of a human suspension 293-F cell line derived ecotropic MLV VPC. Using transposon vector technology, a packaging and envelope expression cassette as well as a transfer vector facilitated the establishment of a stable VPC yielding high titers of up to 5.2 × 106 transducing units/mL (TU/mL). Vectors were concentrated using ultrafiltration devices and upon one freeze-thaw-cycle still routinely yielded titers of > 1 × 106 TU/mL. Formation of replication-competent retroviruses was not detected. However and as a first generation transfer vector was used in this proof-of-concept (POC) study, gag gene sequences were transduced into target cells within a range of 1-10 copies per 1000 genomes indicating the homologous recombination of packaging construct elements with the transfer vector. High yield VPC vector productivity was stable over a couple of months and unintended integration of the transposase gene was not observed. Ecotropic MLV vector particles were demonstrated to efficiently transduce primary murine hematopoietic stem and progenitor cells. This novel concept should foster the future establishment of suspension VPCs.

Cuprizone-induced graded oligodendrocyte vulnerability is regulated by the transcription factor DNA damage-inducible transcript 3.

Oligodendrocytes are integral to efficient neuronal signaling. Loss of myelinating oligodendrocytes is a central feature of many neurological diseases, including multiple sclerosis (MS). The results of neuropathological studies suggest that oligodendrocytes react with differing sensitivity to toxic insults, with some cells dying early during lesion development and some cells being resistant for weeks. This proposed graded vulnerability has never been demonstrated but provides an attractive window for therapeutic interventions. Furthermore, the biochemical pathways associated with graded oligodendrocyte vulnerability have not been well explored. We used immunohistochemistry and serial block-face scanning electron microscopy (3D-SEM) to show that cuprizone-induced metabolic stress results in an "out of phase" degeneration of oligodendrocytes. Although expression induction of stress response transcription factors in oligodendrocytes occurs within days, subsequent oligodendrocyte apoptosis continues for weeks. In line with the idea of an out of phase degeneration of oligodendrocytes, detailed ultrastructural reconstructions of the axon-myelin unit demonstrate demyelination of single internodes. In parallel, genome wide array analyses revealed an active unfolded protein response early after initiation of the cuprizone intoxication. In addition to the cytoprotective pathways, the pro-apoptotic transcription factor DNA damage-inducible transcript 3 (DDIT3) was induced early in oligodendrocytes. In advanced lesions, DDIT3 was as well expressed by activated astrocytes. Toxin-induced oligodendrocyte apoptosis, demyelination, microgliosis, astrocytosis, and acute axonal damage were less intense in the Ddit3-null mutants. This study identifies DDIT3 as an important regulator of graded oligodendrocyte vulnerability in a MS animal model. Interference with this stress cascade might offer a promising therapeutic approach for demyelinating disorders.

Induction of antibacterial proteins and peptides in the coprophilous mushroom Coprinopsis cinerea in response to bacteria.

Bacteria are the main nutritional competitors of saprophytic fungi during colonization of their ecological niches. This competition involves the mutual secretion of antimicrobials that kill or inhibit the growth of the competitor. Over the last years it has been demonstrated that fungi respond to the presence of bacteria with changes of their transcriptome, but the significance of these changes with respect to competition for nutrients is not clear as functional proof of the antibacterial activity of the induced gene products is often lacking. Here, we report the genome-wide transcriptional response of the coprophilous mushroom Coprinopsis cinerea to the bacteria Bacillus subtilis and Escherichia coli. The genes induced upon co-cultivation with each bacterium were highly overlapping, suggesting that the fungus uses a similar arsenal of effectors against Gram-positive and -negative bacteria. Intriguingly, the induced genes appeare to encode predominantly secreted peptides and proteins with predicted antibacterial activities, which was validated by comparative proteomics of the C. cinerea secretome. Induced members of two putative antibacterial peptide and protein families in C. cinerea, the cysteine-stabilized αß-defensins (Csαß-defensins) and the GH24-type lysozymes, were purified, and their antibacterial activity was confirmed. These results provide compelling evidence that fungi are able to recognize the presence of bacteria and respond with the expression of an arsenal of secreted antibacterial peptides and proteins.

Structural Insights into the Mode of Action of the Peptide Antibiotic Copsin.

Defensins make up a class of cysteine-rich antimicrobial peptides, expressed by virtually all eukaryotes as part of their innate immune response. Because of their unique mode of action and rapid killing of pathogenic microbes, defensins are considered promising alternatives to clinically applied antibiotics. Copsin is a defensin-like peptide, previously identified in the mushroom Coprinopsis cinerea. It exerts its activity against a range of Gram-positive bacteria by binding to the peptidoglycan precursor lipid II and prevention of proper cell wall formation. In this study, we present a new workflow for the generation, production, and activity-driven selection of copsin derivatives, based on their expression in Pichia pastoris. One hundred fifty-two single-amino acid mutants and combinations thereof allowed the identification of k-copsin, a peptide variant exhibiting significantly enhanced activity against Bacillus subtilis and Staphylococcus aureus. Furthermore, we performed in silico characterizations of membrane interactions of copsin and k-copsin, in the presence and absence of lipid II. The molecular dynamics data highlighted a high variability in lipid II binding, with a preference for the MurNAc moiety with 47 and 35% of the total contacts for copsin and k-copsin, respectively. Mutated amino acids were located in loop regions of k-copsin and shown to be crucial in the perturbation of the bacterial membrane. These structural studies provide a better understanding of how defensins can be developed toward antibacterial therapies less prone to resistance issues.

Short-term cuprizone feeding induces selective amino acid deprivation with concomitant activation of an integrated stress response in oligodendrocytes.

Cuprizone [bis(cyclohexylidenehydrazide)]-induced toxic demyelination is an experimental approach frequently used to study de- and re-myelination in the central nervous system. In this model, mice are fed with the copper chelator cuprizone which leads to oligodendrocyte apoptosis and subsequent microgliosis, astrocytosis, and demyelination. The underlying mechanisms of cuprizone-induced oligodendrocyte death are still unknown. We analysed differences in amino acid levels after short-term cuprizone exposure (i.e., 4 days). Furthermore, an amino acid response (AAR) pathway activated in oligodendrocytes after cuprizone intoxication was evaluated. Short-term cuprizone exposure resulted in a selective decrease of alanine, glycine, and proline plasma levels, which was paralleled by an increase of apoptotic cells in the liver and a decrease of alanine aminotransferase in the serum. These parameters were paralleled by oligodendrocyte apoptosis and the induction of an AAR with increased expression of the transcription factors ATF-3 and ATF-4 (activating transcription factor-3 and -4). Immunohistochemistry revealed that ATF-3 is exclusively expressed by oligodendrocytes and localized to the nuclear compartment. Our results suggest that cuprizone-induced liver dysfunction results in amino acid starvation and in consequence to the activation of an AAR. We propose that this stress response modulates oligodendrocyte viability in the cuprizone animal model.