Evaluation of two transposases for improving expression of recombinant proteins in Chinese hamster ovary cell stable pools by co-transfection and supertransfection approaches.

Transposons are genetic elements capable of cutting and pasting genes of interest via the action of a transposase and offer many advantages over random or targeted integration of DNA in the creation of Chinese hamster ovary (CHO) cell lines for recombinant protein expression. Unique transposases have different recognition sites, allowing multiple transposases to be co-transfected together. They also allow for supertransfection (transfection on a previously transfected pool or cell line) with a second transposase to integrate additional copies of the same gene or an additional gene without disruption of the previously integrated DNA which to our knowledge has not been previously described in literature. Two fluorescent proteins, EGFP and tagRFP657, were either co-transfected or supertransfected into CHO cells using two unique transposases and showed high expression efficiency with similar expression levels (measured as mean fluorescence intensity), regardless of whether the genes were co-transfected or supertransfected onto an existing stable pool. Additionally, dual selection of the genes, both in the absence of L-glutamine and the presence of puromycin, led to higher expression levels than single selection alone. These results demonstrate that supertransfection using unique transposases could be a useful strategy for increasing titers of existing cell lines or for overexpressing helper (non-therapeutic) genes to improve expression and/or product quality of existing pools and cell lines, potentially saving significant time and resources.

Rescue of a lysosomal storage disorder caused by Grn loss of function with a brain penetrant progranulin biologic.

GRN mutations cause frontotemporal dementia (GRN-FTD) due to deficiency in progranulin (PGRN), a lysosomal and secreted protein with unclear function. Here, we found that Grn-/- mice exhibit a global deficiency in bis(monoacylglycero)phosphate (BMP), an endolysosomal phospholipid we identified as a pH-dependent PGRN interactor as well as a redox-sensitive enhancer of lysosomal proteolysis and lipolysis. Grn-/- brains also showed an age-dependent, secondary storage of glucocerebrosidase substrate glucosylsphingosine. We investigated a protein replacement strategy by engineering protein transport vehicle (PTV):PGRN-a recombinant protein linking PGRN to a modified Fc domain that binds human transferrin receptor for enhanced CNS biodistribution. PTV:PGRN rescued various Grn-/- phenotypes in primary murine macrophages and human iPSC-derived microglia, including oxidative stress, lysosomal dysfunction, and endomembrane damage. Peripherally delivered PTV:PGRN corrected levels of BMP, glucosylsphingosine, and disease pathology in Grn-/- CNS, including microgliosis, lipofuscinosis, and neuronal damage. PTV:PGRN thus represents a potential biotherapeutic for GRN-FTD.

TREM2 Regulates Microglial Cholesterol Metabolism upon Chronic Phagocytic Challenge.

Loss-of-function (LOF) variants of TREM2, an immune receptor expressed in microglia, increase Alzheimer's disease risk. TREM2 senses lipids and mediates myelin phagocytosis, but its role in microglial lipid metabolism is unknown. Combining chronic demyelination paradigms and cell sorting with RNA sequencing and lipidomics, we find that wild-type microglia acquire a disease-associated transcriptional state, while TREM2-deficient microglia remain largely homeostatic, leading to neuronal damage. TREM2-deficient microglia phagocytose myelin debris but fail to clear myelin cholesterol, resulting in cholesteryl ester (CE) accumulation. CE increase is also observed in APOE-deficient glial cells, reflecting impaired brain cholesterol transport. This finding replicates in myelin-treated TREM2-deficient murine macrophages and human iPSC-derived microglia, where it is rescued by an ACAT1 inhibitor and LXR agonist. Our studies identify TREM2 as a key transcriptional regulator of cholesterol transport and metabolism under conditions of chronic myelin phagocytic activity, as TREM2 LOF causes pathogenic lipid accumulation in microglia.

Phenotypic and molecular characterization of invasive serogroup W135 Neisseria meningitidis strains from 1990 to 2005 in Brazil.

OBJECTIVE: Neisseria meningitidis serogroup W135 has been associated with global outbreaks since the 2000 Hajj. Considering that N. meningitidis serogroup W135 is the third most prevalent serogroup isolated in Brazil in the last 10 years, and the possibility that the Hajj-related N. meningitidis serogroup W135 clone has been causing disease in Brazil, the present study characterized invasive N. meningitidis serogroup W135 isolates recovered in Brazil from 1990 to 2005. METHODS: The isolates were characterized by serotyping, PorA and PorB VR typing, FetA and 16S rRNA typing, multilocus sequence typing (MLST) and pulsed field gel electrophoresis (PFGE). RESULTS: Based on MLST, 73% of the isolates were clustered in one major clone of ST-11 complex/ET37 complex. Strains of this clone had the same STs, serotypes and PorA VR types as found in Hajj-related N. meningitidis serogroup W135 clone. One of these strains had the Hajj-2000 outbreak strain genotype, including 16S rRNA gene sequence 31 and 84% relatedness by PFGE. CONCLUSION: Taken together, these data suggest that the Hajj-related N. meningitidis serogroup W135 clone is present in Brazil but has not yet caused a substantial number of infections. Given the emergence of N. meningitidis serogroup W135 globally and the unpredictability of meningococcal disease epidemiology, continued surveillance for this invasive N. meningitidis serogroup W135 clone is needed for control and prevention strategies.