Primary T-cell-based delivery platform for in vivo synthesis of engineered proteins.

Primary T cell has been transformed into a cell-based delivery platform that synthesizes complex biologics at the disease site with spatiotemporal resolution. This broadly applicable technology can circumvent toxicities due to systemic administration of biologics that necessitates the use of high doses and may diffuse to the healthy tissues. Its clinical translation, however, has been impeded by manufacturing bottlenecks. In this work, a range of process parameters were investigated for increasing the production yield of the primary T cells engineered for delivery function. Compared to the common spinoculation-based method, the transduction yield was enhanced ~2.5-fold by restricting the transduction reaction volume for maximizing the lentivector-to-T-cell contact. Cell density and cytokines used in the expansion process were adjusted to achieve >100-fold expansion of the T-cell-based delivery platform in 14 days, and the function of these cells was validated in vivo using intraperitoneally implanted tumor cells. The primary T-cell-based delivery platform has human applications because it can be scaled and administrated to express a broad range of therapeutic proteins (e.g., cytokines, interferons, enzymes, agonists, and antagonists) at the disease site, obviating the need for systemic delivery of large doses of these proteins.

Auxilin-dynamin interactions link the uncoating ATPase chaperone machinery with vesicle formation.

The large GTPase dynamin is required for budding of clathrin-coated vesicles from the plasma membrane, after which the clathrin coat is removed by the chaperone Hsc70 and its cochaperone auxilin. Recent evidence suggests that the GTP-bound form of dynamin may recruit factors that execute the fission reaction. Here, we show that dynamin:GTP binds to Hsc70 and auxilin. We mapped two domains within auxilin that interact with dynamin, and these domains inhibit endocytosis when overexpressed in HeLa cells or when added in a permeable cell assay. The inhibition is not due to impairment of clathrin uncoating or to altered clathrin distribution in cells. Thus, in addition to its requirement for clathrin uncoating, our results show that auxilin also acts during the early steps of clathrin-coated vesicle formation. The data suggest that dynamin regulates the action of molecular chaperones in vesicle budding during endocytosis.

Hsc70 is required for endocytosis and clathrin function in Drosophila.

By screening for Drosophila mutants exhibiting aberrant bride of sevenless (Boss) staining patterns on eye imaginal disc epithelia, we have recovered a point mutation in Hsc70-4, the closest homologue to bovine clathrin uncoating ATPase. Although the mutant allele was lethal, analysis of mutant clones generated by FLP/FRT recombination demonstrated that the Sevenless-mediated internalization of Boss was blocked in mutant Hsc70-4 eye disc epithelial cells. Endocytosis of other probes was also greatly inhibited in larval Garland cells. Immunostaining and EM analysis of the mutant cells revealed disruptions in the organization of endosomal/lysosomal compartments, including a substantial reduction in the number of clathrin-coated structures in Garland cells. The Hsc70-4 mutation also interacted genetically with a dominant-negative mutant of dynamin, a gene required for the budding of clathrin-coated vesicles (CCVs). Consistent with these phenotypes, recombinant mutant Hsc70 proteins exhibited diminished clathrin uncoating activity in vitro. Together, these data provide genetic support for the long-suspected role of Hsc70 in clathrin-mediated endocytosis, at least in part by inhibiting the uncoating of CCVs.

Assays and functional properties of auxilin-dynamin interactions.

The large GTPase dynamin is required for budding of clathrin-coated vesicles from the plasma membrane, but its mechanism of action is still not understood. Growing evidence indicates that the GTP-bound form of dynamin recruits downstream partners that execute the fission reaction. Recently, we reported nucleotide-dependent interactions between dynamin and auxilin, which suggested that auxilin cooperates with dynamin during vesicle formation. Here we describe three different in vitro assays that monitor auxilin-dynamin interactions, as well as fluorescence lifetime imaging microscopy that identify direct interactions between dynamin and auxilin in cells.

Uncoating ATPase Hsc70 is recruited by invariant chain and controls the size of endocytic compartments.

Targeting of class II major histocompatibility complex molecules to endocytic compartments is mediated by their association with the invariant chain (Ii). Although the identity of certain sorting signals located in Ii's cytoplasmic tail is known, proteins that interact with Ii's cytoplasmic tail in living cells remain to be identified. Synthesis of a biotinylated trimeric Ii cytoplasmic tail allowed the retrieval of two proteins that interact with this domain. We identify one of them as the 70-kDa heat-shock cognate protein (hsc70), the uncoating ATPase of clathrin-coated vesicles, and the other as its mitochondrial homologue, the glucose-regulated protein grp75. Expression of Ii in COS cells results in the formation of large endocytic compartments. We observe extensive colocalization of hsc70 with Ii in these macrosomes. Expression of a dominant-negative (K71M) green fluorescent protein-tagged version of hsc70 counteracted the ability of Ii to modify the endocytic pathway, demonstrating an interaction in vivo of Ii with hsc70 as part of the machinery responsible for macrosome formation.