Accurate definition of control strategies using cross validated stepwise regression and Monte Carlo simulation.

Drug manufacturing processes must consistently deliver safe and effective product. A key part of achieving this is process validation utilizing Quality by Design (QbD) principles. To meet process validation requirements, process characterization (PC) studies are often performed to expand process understanding and establish an appropriate control strategy that enables the manufacturing process to consistently deliver a target product profile.

Highly efficient deletion of FUT8 in CHO cell lines using zinc-finger nucleases yields cells that produce completely nonfucosylated antibodies.

IgG1 antibodies produced in Chinese hamster ovary (CHO) cells are heavily alpha1,6-fucosylated, a modification that reduces antibody-dependent cellular cytotoxicity (ADCC) and can inhibit therapeutic antibody function in vivo. Addition of fucose is catalyzed by Fut8, a alpha1,6-fucosyltransferase. FUT8(-/-) CHO cell lines produce completely nonfucosylated antibodies, but the difficulty of recapitulating the knockout in protein-production cell lines has prevented the widespread adoption of FUT8(-/-) cells as hosts for antibody production.

Histone deimination antagonizes arginine methylation.

Methylation of arginine residues within histone H3 has been linked to active transcription. This modification appears on the estrogen-regulated pS2 promoter when the CARM1 methyltransferase is recruited during transcriptional activation. Here we describe a process, deimination, that converts histone arginine to citrulline and antagonizes arginine methylation.

Repression of vascular endothelial growth factor A in glioblastoma cells using engineered zinc finger transcription factors.

Angiogenic factors are necessary for tumor proliferation and thus are attractive therapeutic targets. In this study, we have used engineered zinc finger protein (ZFP) transcription factors (TFs) to repress expression of vascular endothelial growth factor (VEGF)-A in human cancer cell lines. We create potent transcriptional repressors by fusing a designed ZFP targeted to the VEGF-A promoter with either the ligand-binding domain of thyroid hormone receptor alpha or its viral relative, vErbA.

Zinc-finger protein-targeted gene regulation: genomewide single-gene specificity.

Zinc-finger protein transcription factors (ZFP TFs) can be designed to control the expression of any desired target gene, and thus provide potential therapeutic tools for the study and treatment of disease. Here we report that a ZFP TF can repress target gene expression with single-gene specificity within the human genome. A ZFP TF repressor that binds an 18-bp recognition sequence within the promoter of the endogenous CHK2 gene gives a >10-fold reduction in CHK2 mRNA and protein.

P300 transcriptional repression is mediated by SUMO modification.

p300 and CREB binding protein can both activate and repress transcription. Here, we locate the CRD1 transcriptional repression domain between residues 1017 and 1029 of p300. This region contains two copies of the sequence psiKxE that are modified by the ubiquitin-like protein SUMO-1.

Gene-specific targeting of H3K9 methylation is sufficient for initiating repression in vivo.

Covalent modifications of chromatin have emerged as key determinants of the genome's transcriptional competence. Histone H3 lysine 9 (H3K9) methylation is an epigenetic signal that is recognized by HP1 and correlates with gene silencing in a variety of organisms. Discovery of the enzymes that catalyze H3K9 methylation has identified a second gene-specific function for this modification in transcriptional repression.

Induction of transcription through the p300 CRD1 motif by p21WAF1/CIP1 is core promoter specific and cyclin dependent kinase independent.

The tumor suppressors p300 and CREB-binding protein (CBP) are both multifunctional transcriptional coactivators. We have previously found that the cyclin dependent kinase (CDK) inhibitor p21(WAF1/CIP1) can stimulate transactivation by p300 and CBP through inhibiting transcriptional repression by a discrete domain within these proteins termed CRD1. Given the large number of p300/CBP associated functions, it is unlikely that p21 regulates the expression of every gene under their control, however.