Generation of FX and Gmds CHOZN host cell lines for the production of afucosylated therapeutic antibodies.

Antibody-dependent cellular cytotoxicity (ADCC) is the primary mechanism of actions for several marketed therapeutic antibodies (mAbs) and for many more in clinical trials. The ADCC efficacy is highly dependent on the ability of therapeutic mAbs to recruit effector cells such as natural killer cells, which induce the apoptosis of targeted cells. The recruitment of effector cells by mAbs is negatively affected by fucose modification of N-Glycans on the Fc; thus, utilization of afucosylated mAbs has been a trend for enhanced ADCC therapeutics.

Lentiviral arrays for live-cell dynamic monitoring of gene and pathway activity during stem cell differentiation.

Uncovering the complexity of mesenchymal stem cell (MSC) differentiation requires novel methods to capture the dynamics of the process in a quantitative and high-throughput manner. To this end, we developed a lentiviral array (LVA) of reporters to capture the dynamics of gene and pathway activity during MSC differentiation into adipogenic, chondrogenic, and osteogenic lineages. Our results identified signature promoters and pathways with unique activation profile for each MSC lineage.

Adherens junction formation inhibits lentivirus entry and gene transfer.

Although cellular signaling pathways that affect lentivirus infection have been investigated, the role of cell-cell interactions in lentiviral gene delivery remains elusive. In the course of our studies we observed that lentiviral gene transfer was a strong function of the position of epithelial cells within colonies. While peripheral cells were transduced efficiently, cells in the center of colonies were resistant to gene transfer.

JNK regulates compliance-induced adherens junctions formation in epithelial cells and tissues.

We demonstrate that c-Jun N-terminal kinase (JNK) responds to substrate stiffness and regulates adherens junction (AJ) formation in epithelial cells in 2D cultures and in 3D tissues in vitro and in vivo. Rigid substrates led to JNK activation and AJ disassembly, whereas soft matrices suppressed JNK activity leading to AJ formation. Expression of constitutively active JNK (MKK7-JNK1) induced AJ dissolution even on soft substrates, whereas JNK knockdown (using shJNK) induced AJ formation even on hard substrates.

Engineering fibrinogen-binding VSV-G envelope for spatially- and cell-controlled lentivirus delivery through fibrin hydrogels.

We recently demonstrated that fibrin hydrogels can be used as vehicles for efficient lentivirus gene delivery. Gene transfer in fibrin gels was strongly dependent on matrix degradation by target cells but a fraction of lentiviral particles diffused out of the gels over time compromising spatial control of gene transfer. To overcome this challenge, we engineered lentiviral particles that bind covalently to fibrin during polymerization.

JNK1 is required for lentivirus entry and gene transfer.

Although a lot of progress has been made in development of lentiviral vectors for gene therapy, the interactions of these vectors with cellular factors have not been explored adequately. Here we show that lentivirus infection phosphorylates JNK and that blocking the kinase activity of JNK decreases gene transfer in a dose-dependent manner, regardless of the viral envelope glycoprotein. Knockdown by small interfering RNA (siRNA) revealed that JNK1 but not JNK2 was required for productive gene transfer.

JNK regulates binding of alpha-catenin to adherens junctions and cell-cell adhesion.

We recently reported that c-Jun N-terminal kinase (JNK) is associated with adherens junctions and phosphorylates β-catenin at serine 33/37 and threonine 41. Here, we report that inhibition of JNK led to formation of adherens junctions, which was accompanied by dissociation of α-catenin from the β-catenin/E-cadherin complex and increased association of α-catenin with the cytoskeleton. Conversely, activation of JNK increased binding of α-catenin to β-catenin, which was blocked by the JNK inhibitor SP600125 or JNK siRNA.

Fibrin-mediated lentivirus gene transfer: implications for lentivirus microarrays.

We employed fibrin hydrogel as a bioactive matrix for lentivirus mediated gene transfer. Fibrin-mediated gene transfer was highly efficient and exhibited strong dependence on fibrinogen concentration. Efficient gene transfer was achieved with fibrinogen concentration between 3.

Cell-controlled and spatially arrayed gene delivery from fibrin hydrogels.

We investigated fibrin-mediated gene transfer by embedding pDNA within the hydrogel during polymerization and using two modes of gene transfection with cells placed either on the surface (2D transfection) or within the hydrogel (3D transfection). Using this model, we found that cell transfection depended strongly on the local cell-pDNA microenvironment as defined by the 2D vs. 3D context, target cell type and density, as well as fibrinogen and pDNA concentrations.