Identification of infectious viruses for risk-based virus testing of CHO unprocessed bulk using next-generation sequencing.

It is important to increase manufacturing speed to make medicines more widely available. One bottleneck for CHO-based drug substance release is the in vitro viral (IVV) cell-based assay on unprocessed bulk. To increase process speed, we evaluate the suitability of replacing the IVV cell-based assay with next-generation sequencing (NGS). First, we outline how NGS is currently used in the pharmaceutical industry, and how it may apply to CHO virus testing. Second, we examine CHO virus contamination history. Since prior virus contaminants can replicate in the production bioreactor, we perform a literature search and classify 159 viruses as high, medium, low, or unknown risk based on their ability to infect CHO cells. Overall, the risk of virus contamination during the CHO manufacturing process is low. Only six viruses were reported to have contaminated CHO bioprocesses over the past several decades, and were primarily caused by fetal bovine serum or cell culture components. These virus contamination events can be mitigated through limitation and control of raw materials, combined with virus testing and virus clearance technologies. The list of CHO infectious viruses provides a starting framework for virus safety risk assessment and NGS development. Furthermore, ICH Q5A (R2) includes NGS as a molecular method for adventitious agent testing, paving a path forward for modernizing CHO virus testing.

Retrotransposons in embryogenesis and neurodevelopment.

Retrotransposable elements (RTEs) are genetic elements that can replicate and insert new copies into different genomic locations. RTEs have long been identified as 'parasitic genes', as their mobilization can cause mutations, DNA damage, and inflammation. Interestingly, high levels of retrotransposon activation are observed in early embryogenesis and neurodevelopment, suggesting that RTEs may possess functional roles during these stages of development. Recent studies demonstrate that RTEs can function as transcriptional regulatory elements through mechanisms such as chromatin organization and noncoding RNAs. It is clear, however, that RTE expression and activity must be restrained at some level during development, since overactivation of RTEs during neurodevelopment is associated with several developmental disorders. Further investigation is needed to understand the importance of RTE expression and activity during neurodevelopment and the balance between RTE-regulated development and RTE-mediated pathogenesis.

Condensin-mediated restriction of retrotransposable elements facilitates brain development in Drosophila melanogaster.

Neural stem and progenitor cell (NSPC) maintenance is essential for ensuring that organisms are born with proper brain volumes and head sizes. Microcephaly is a disorder in which babies are born with significantly smaller head sizes and cortical volumes. Mutations in subunits of the DNA organizing complex condensin have been identified in microcephaly patients. However, the molecular mechanisms by which condensin insufficiency causes microcephaly remain elusive. We previously identified conserved roles for condensins in repression of retrotransposable elements (RTEs). Here, we show that condensin subunit knockdown in NSPCs of the Drosophila larval central brain increases RTE expression and mobility which causes cell death, and significantly decreases adult head sizes and brain volumes. These findings suggest that unrestricted RTE expression and activity may lead to improper brain development in condensin insufficient organisms, and lay the foundation for future exploration of causative roles for RTEs in other microcephaly models.

Distinct requirements for Tcf3 and Tcf12 during oligodendrocyte development in the mouse telencephalon.

BACKGROUND: E-proteins encoded by Tcf3, Tcf4, and Tcf12 are class I basic helix-loop-helix (bHLH) transcription factors (TFs) that are thought to be widely expressed during development. However, their function in the developing brain, specifically in the telencephalon remains an active area of research. Our study examines for the first time if combined loss of two E-proteins (Tcf3 and Tcf12) influence distinct cell fates and oligodendrocyte development in the mouse telencephalon. METHODS: We generated Tcf3/12 double conditional knockouts (dcKOs) using Olig2Cre/+ or Olig1Cre/+ to overcome compensatory mechanisms between E-proteins and to understand the specific requirement for Tcf3 and Tcf12 in the ventral telencephalon and during oligodendrogenesis. We utilized a combination of in situ hybridization, immunohistochemistry, and immunofluorescence to address development of the telencephalon and oligodendrogenesis at embryonic and postnatal stages in Tcf3/12 dcKOs. RESULTS: We show that the E-proteins Tcf3 and Tcf12 are expressed in progenitors of the embryonic telencephalon and throughout the oligodendrocyte lineage in the postnatal brain. Tcf3/12 dcKOs showed transient defects in progenitor cells with an enlarged medial ganglionic eminence (MGE) region which correlated with reduced generation of embryonic oligodendrocyte progenitor cells (OPCs) and increased expression of MGE interneuron genes. Postnatal Tcf3/12 dcKOs showed a recovery of OPCs but displayed a sustained reduction in mature oligodendrocytes (OLs). Interestingly, Tcf4 remained expressed in the dcKOs suggesting that it cannot compensate for the loss of Tcf3 and Tcf12. Generation of Tcf3/12 dcKOs with Olig1Cre/+ avoided the MGE morphology defect caused by Olig2Cre/+ but dcKOs still exhibited reduced embryonic OPCs and subsequent reduction in postnatal OLs. CONCLUSION: Our data reveal that Tcf3 and Tcf12 play a role in controlling OPC versus cortical interneuron cell fate decisions in MGE progenitors in addition to playing roles in the generation of embryonic OPCs and differentiation of postnatal OLs in the oligodendrocyte lineage.

Generation of a Mouse Model to Study the Noonan Syndrome Gene Lztr1 in the Telencephalon.

The leucine zipper-like transcriptional regulator 1 (Lztr1) is a BTB-Kelch domain protein involved in RAS/MAPK pathway regulation. Mutations in LZTR1 are associated with cancers and Noonan syndrome, the most common RASopathy. The expression and function of Lztr1 in the developing brain remains poorly understood. Here we show that Lztr1 is expressed in distinct regions of the telencephalon, the most anterior region of the forebrain. Lztr1 expression was robust in the cortex, amygdala, hippocampus, and oligodendrocytes in the white matter. To gain insight into the impact of Lztr1 deficiency, we generated a conditional knockout (cKO) restricted to the telencephalon using Foxg1 IREScre/+. Lztr1 cKOs are viable to postnatal stages and show reduced Lztr1 expression in the telencephalon. Interestingly, Lztr1 cKOs exhibit an increase in MAPK pathway activation in white matter regions and subsequently show an altered expression of stage-specific markers in the oligodendrocyte lineage with increased oligodendrocyte progenitor cells (OPCs) and decreased markers of oligodendrocyte differentiation. Moreover, Lztr1 cKOs also exhibit an increased expression of the astrocyte marker GFAP. These results highlight the generation of a new mouse model to study Lztr1 deficiency in the brain and reveal a novel role for Lztr1 in normal oligodendrocyte and astrocyte development in the telencephalon.

A role for sustained MAPK activity in the mouse ventral telencephalon.

The MAPK pathway is a major growth signal that has been implicated during the development of progenitors, neurons, and glia in the embryonic brain. Here, we show that the MAPK pathway plays an important role in the generation of distinct cell types from progenitors in the ventral telencephalon. Our data reveal that phospho-p44/42 (called p-ERK1/2) and the ETS transcription factor Etv5, both downstream effectors in the MAPK pathway, show a regional bias in expression during ventral telencephalic development, with enriched expression in the dorsal region of the LGE and ventral region of the MGE at E13.5 and E15.5. Interestingly, expression of both factors becomes more uniform in ventricular zone (VZ) progenitors by E18.5. To gain insight into the role of MAPK activity during progenitor cell development, we used a cre inducible constitutively active MEK1 allele (RosaMEK1DD/+) in combination with a ventral telencephalon enriched cre (Gsx2e-cre) or a dorsal telencephalon enriched cre (Emx1cre/+). Sustained MEK/MAPK activity in the ventral telencephalon (Gsx2e-cre; RosaMEK1DD/+) expanded dorsal lateral ganglionic eminence (dLGE) enriched genes (Gsx2 and Sp8) and oligodendrocyte progenitor cell (OPC) markers (Olig2, Pdgfrα, and Sox10), and also reduced markers in the ventral (v) LGE domain (Isl1 and Foxp1). Activation of MEK/MAPK activity in the dorsal telencephalon (Emx1cre/+; RosaMEK1DD/+) did not initially activate the expression of dLGE or OPC genes at E15.5 but ectopic expression of Gsx2 and OPC markers were observed at E18.5. These results support the idea that MAPK activity as readout by p-ERK1/2 and Etv5 expression is enriched in distinct subdomains of ventral telencephalic progenitors during development. In addition, sustained activation of the MEK/MAPK pathway in the ventral or dorsal telencephalon influences dLGE and OPC identity from progenitors.