Disruption of diphthamide synthesis genes and resulting toxin resistance as a robust technology for quantifying and optimizing CRISPR/Cas9-mediated gene editing.

We have devised an effective and robust method for the characterization of gene-editing events. The efficacy of editing-mediated mono- and bi-allelic gene inactivation and integration events is quantified based on colony counts. The combination of diphtheria toxin (DT) and puromycin (PM) selection enables analyses of 10,000-100,000 individual cells, assessing hundreds of clones with inactivated genes per experiment. Mono- and bi-allelic gene inactivation is differentiated by DT resistance, which occurs only upon bi-allelic inactivation. PM resistance indicates integration. The robustness and generalizability of the method were demonstrated by quantifying the frequency of gene inactivation and cassette integration under different editing approaches: CRISPR/Cas9-mediated complete inactivation was ~30-50-fold more frequent than cassette integration. Mono-allelic inactivation without integration occurred >100-fold more frequently than integration. Assessment of gRNA length confirmed 20mers to be most effective length for inactivation, while 16-18mers provided the highest overall integration efficacy. The overall efficacy was ~2-fold higher for CRISPR/Cas9 than for zinc-finger nuclease and was significantly increased upon modulation of non-homologous end joining or homology-directed repair. The frequencies and ratios of editing events were similar for two different DPH genes (independent of the target sequence or chromosomal location), which indicates that the optimization parameters identified with this method can be generalized.

Loss of diphthamide pre-activates NF-κB and death receptor pathways and renders MCF7 cells hypersensitive to tumor necrosis factor.

The diphthamide on human eukaryotic translation elongation factor 2 (eEF2) is the target of ADP ribosylating diphtheria toxin (DT) and Pseudomonas exotoxin A (PE). This modification is synthesized by seven dipthamide biosynthesis proteins (DPH1-DPH7) and is conserved among eukaryotes and archaea. We generated MCF7 breast cancer cell line-derived DPH gene knockout (ko) cells to assess the impact of complete or partial inactivation on diphthamide synthesis and toxin sensitivity, and to address the biological consequence of diphthamide deficiency. Cells with heterozygous gene inactivation still contained predominantly diphthamide-modified eEF2 and were as sensitive to PE and DT as parent cells. Thus, DPH gene copy number reduction does not affect overall diphthamide synthesis and toxin sensitivity. Complete inactivation of DPH1, DPH2, DPH4, and DPH5 generated viable cells without diphthamide. DPH1ko, DPH2ko, and DPH4ko harbored unmodified eEF2 and DPH5ko ACP- (diphthine-precursor) modified eEF2. Loss of diphthamide prevented ADP ribosylation of eEF2, rendered cells resistant to PE and DT, but does not affect sensitivity toward other protein synthesis inhibitors, such as saporin or cycloheximide. Surprisingly, cells without diphthamide (independent of which the DPH gene compromised) were presensitized toward nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) and death-receptor pathways without crossing lethal thresholds. In consequence, loss of diphthamide rendered cells hypersensitive toward TNF-mediated apoptosis. This finding suggests a role of diphthamide in modulating NF-κB, death receptor, or apoptosis pathways.

Bispecific antibody derivatives with restricted binding functionalities that are activated by proteolytic processing.

We have designed bispecific antibodies that bind one target (anti-Her3) in a bivalent IgG-like manner and contain one additional binding entity (anti-cMet) composed of one V(H) and one V(L) domain connected by a disulfide bond. The molecules are assembled by fusing a V(H,Cys44) domain via flexible connector peptides to the C-terminus of one H-chain (heavy chain), and a V(L,Cys100) to another H-chain. To ensure heterodimerization during expression in mammalian cells, we introduced complementary knobs-into-holes mutations into the different H-chains. The IgG-shaped trivalent molecules carry as third binding entity one disulfide-stabilized Fv (dsFv) without a linker between V(H) and V(L). Tethering the V(H) and V(L) domains at the C-terminus of the C(H)3 domain decreases the on-rates of the dsFv to target antigens without affecting off-rates. Steric hindrance resolves upon removal of one side of the double connection by proteolysis: this improves flexibility and accessibility of the dsFv and fully restores antigen access and affinity. This technology has multiple applications: (i) in cases where single-chain linkers are not desired, dsFvs without linkers can be generated by addition of furin site(s) in the connector that are processed during expression within mammalian cells; (ii) highly active (toxic) entities which affect expression can be produced as inactive dsFvs and subsequently be activated (e.g. via PreScission cleavage) during purification; (iii) entities can be generated which are targeted by the unrestricted binding entity and can be activated by proteases in target tissues. For example, Her3-binding molecules containing linkers with recognition sequences for matrix metalloproteases or urokinase, whose inactivated cMet binding site is activated by proteolytic processing.

Bispecific antibody derivatives based on full-length IgG formats.

Monoclonal antibodies have emerged as an effective therapeutic modality, and numerous antibodies have been approved for the treatment of several severe diseases or are currently in clinical development. To improve their therapeutic potential, monoclonal antibodies are constantly evolved by protein engineering. Particularly, the generation of bispecific antibodies raised special interest because of their ability to bind two different antigens at the same time, and the efficiency of these formats has been demonstrated in several clinical and preclinical studies. Up to now, the major drawbacks in using bispecific antibodies as a therapeutic agent have been difficult design and low-yield expression of homogeneous antibody populations. However, major technological improvements were made in protein engineering during the last years. This allows the design of several new IgG-based bispecific antibody formats that can be prepared in high yields and high homogeneity using conventional expression and purification techniques. Especially, recent development of IgG-fusions with disulfide-stabilized Fv fragments and of CrossMab-technologies facilitates the generation of bispecific antibodies with IgG-like architectures. Here we describe design principles and methods to express and purify different bispecific antibody formats derived from full-length IgGs.

Generation of fluorescent IgG fusion proteins in mammalian cells.

The generation of recombinantly produced fluorescent antibody derivatives that are derived from full-length immunoglobulin G (IgG) has until now been problematic. One major reason for that lies in different and partially incompatible secretion- and folding-requirements of antibodies and green fluorescent protein (GFP) derived fluorescent entities in mammalian cells. The use of citrine as fluorescent fusion entity can overcome this limitation. Citrine is a modified yellow fluorescent protein (YFP) derivative which in contrast to GFP and yellow fluorescent protein (YFP) folds effectively and properly in the endoplasmic reticulum (ER) of mammalian cells. Provided that proper design parameters regarding fusion positions and linker/connector sequences are applied, citrine can be fused to different positions of IgGs and be expressed without interfering with secretion capability or functionality of IgG-citrine derivatives. Because IgG-citrine fusions are stable and retain biophysical properties of IgGs, they can be expressed and purified in the same manner as regular antibodies. IgG-citrine fusions not only retain the binding properties (affinity and specificity) of antibodies but also contain Fc-regions (useful for immunoassay applications), and are fully defined molecules (in contrast to antibody conjugates with fluorophores).

Human-protein-derived peptides for intracellular delivery of biomolecules.

Access of therapeutic biomolecules to cytoplasmic and nuclear targets is hampered by the inability of these molecules to cross biological membranes. Approaches to overcome this hurdle involve CPPs (cell-penetrating peptides) or protein transduction domains. Most of these require rather high concentrations to elicit cell-penetrating functionality, are non-human, pathogen-derived or synthetic entities, and may therefore not be tolerated or even immunogenic. We identified novel human-protein-derived CPPs by a combination of in silico and experimental analyses: polycationic CPP candidates were identified in an in silico library of all 30-mer peptides of the human proteome. Of these peptides, 60 derived from extracellular proteins were evaluated experimentally. Cell viability and siRNA (small interfering RNA) transfection assays revealed that 20 out of the 60 peptides were functional. Three of these showed CPP functionality without interfering with cell viability. A peptide derived from human NRTN (neurturin), which contains an α-helix, performed the best in our screen and was uniformly taken up by cultured cells. Examples for payloads that can be delivered to the cytosol by the NRTN peptide include complexed siRNAs and both N- and C-terminally fused pro-apoptotic peptides.

Bispecific digoxigenin-binding antibodies for targeted payload delivery.

Bispecific antibodies that bind cell-surface targets as well as digoxigenin (Dig) were generated for targeted payload delivery. Targeting moieties are IgGs that bind the tumor antigens Her2, IGF1R, CD22, or LeY. A Dig-binding single-chain Fv was attached in disulfide-stabilized form to C termini of CH3 domains of targeting antibodies. Bispecific molecules were expressed in mammalian cells and purified in the same manner as unmodified IgGs. They are stable without aggregation propensity and retain binding specificity/affinity to cell-surface antigens and Dig. Digoxigeninylated payloads were generated that retain full functionality and can be complexed to bispecific antibodies in a defined 21 ratio. Payloads include small compounds (Dig-Cy5, Dig-Doxorubicin) and proteins (Dig-GFP). Complexed payloads are targeted by the bispecifics to cancer cells and because these complexes are stable in serum, they can be applied for targeted delivery. Because Dig bispecifics also effectively capture digoxigeninylated compounds under physiological conditions, separate administration of uncharged Dig bispecifics followed by application of Dig payload is sufficient to achieve antibody-mediated targeting in vitro and in vivo.

Fluorescent Citrine-IgG fusion proteins produced in mammalian cells.

Genetically encoded fluorescent antibodies are desirable for many applications in biotechnology, proteomics, microscopy, cell biology and molecular diagnostics, although efficient production of fluorescent IgGs in mammalian cells has been hampered by different and mutually incompatible secretion- and folding-requirements of antibodies and green fluorescent protein-derived fluorescent entities. Here, we show that this hurdle can be overcome by generating whole antibody fusions with Citrine, a modified yellow fluorescent protein that folds properly in the endoplasmic reticulum of mammalian cells. Applying optimized connector sequences, one or more Citrine molecules can be fused to different positions of IgGs without interfering with folding, secretion or function of the fusion proteins. These proteins can be transiently expressed and purified to similar yields as unmodified antibodies using standard technologies. IgG-Citrine fusions fully retain binding specificity and affinity, and can be applied to assays that require labeled IgG. A particularly interesting feature is the pH-dependency of Citrine fluorescence. This makes IgG-Citrine fusion proteins a valuable tool to track antibody target binding, internalization and subsequent intracellular trafficking to acidic compartments.

Multiple Rab GTPase binding sites in GCC185 suggest a model for vesicle tethering at the trans-Golgi.

GCC185, a trans-Golgi network-localized protein predicted to assume a long, coiled-coil structure, is required for Rab9-dependent recycling of mannose 6-phosphate receptors (MPRs) to the Golgi and for microtubule nucleation at the Golgi via CLASP proteins. GCC185 localizes to the Golgi by cooperative interaction with Rab6 and Arl1 GTPases at adjacent sites near its C terminus. We show here by yeast two-hybrid and direct biochemical tests that GCC185 contains at least four additional binding sites for as many as 14 different Rab GTPases across its entire length. A central coiled-coil domain contains a specific Rab9 binding site, and functional assays indicate that this domain is important for MPR recycling to the Golgi complex. N-Terminal coiled-coils are also required for GCC185 function as determined by plasmid rescue after GCC185 depletion by using small interfering RNA in cultured cells. Golgi-Rab binding sites may permit GCC185 to contribute to stacking and lateral interactions of Golgi cisternae as well as help it function as a vesicle tether.

Analysis of Rab GTPase and GTPase-activating protein function at primary cilia.

Primary cilia are sensory structures on the cell surface whose formation requires tight integration of microtubules and polarized membrane trafficking events. Rabs are GTP-binding proteins of the Ras superfamily that control directed membrane trafficking by regulating membrane-membrane and membrane-cytoskeleton interactions. This chapter describes cell biological and biochemical methods for the analysis of Rab function and Rab GTPase-activating proteins during primary cilia formation.

Analysis of GTPase-activating proteins: Rab1 and Rab43 are key Rabs required to maintain a functional Golgi complex in human cells.

Rab GTPases control vesicle movement and tethering membrane events in membrane trafficking. We used the 38 human Rab GTPase activating proteins (GAPs) to identify which of the 60 Rabs encoded in the human genome function at the Golgi complex. Surprisingly, this screen identified only two GAPs, RN-tre and TBC1D20, disrupting both Golgi organization and protein transport. RN-tre is the GAP for Rab43, and controls retrograde transport into the Golgi from the endocytic pathway. TBC1D20 is the ER-localized GAP for Rab1, and is the only GAP blocking the delivery of secretory cargo from the ER to the cell surface. Strikingly, its expression causes the loss of the Golgi complex, highlighting the importance of Rab1 for Golgi biogenesis. These effects can be antagonized by reticulon, a binding partner for TBC1D20 in the ER. Together, these findings indicate that Rab1 and Rab43 are key Rabs required for the biogenesis and maintenance of a functional Golgi structure, and suggest that other Rabs acting at the Golgi complex are likely to be functionally redundant.

Functional dissection of Rab GTPases involved in primary cilium formation.

Primary cilia are sensory structures involved in morphogen signalling during development, liquid flow in the kidney, mechanosensation, sight, and smell (Badano, J.L., N. Mitsuma, P.L. Beales, and N. Katsanis. 2006. Annu. Rev. Genomics Hum. Genet. 7:125-148; Singla, V., and J.F. Reiter. 2006. Science. 313:629-633.). Mutations that affect primary cilia are responsible for several diseases, including neural tube defects, polycystic kidney disease, retinal degeneration, and cancers (Badano et al., 2006; Singla and Reiter, 2006). Primary cilia formation and function requires tight integration of the microtubule cytoskeleton with membrane trafficking (Singla and Reiter, 2006), and this is poorly understood. We show that the Rab GTPase membrane trafficking regulators Rab8a, -17, and -23, and their cognate GTPase-activating proteins (GAPs), XM_037557, TBC1D7, and EVI5like, are involved in primary cilia formation. However, other human Rabs and GAPs are not. Additionally, Rab8a specifically interacts with cenexin/ODF2, a basal body and microtubule binding protein required for cilium biogenesis (Ishikawa, H., A. Kubo, S. Tsukita, and S. Tsukita. 2005. Nat. Cell Biol. 7:517-524), and is the sole Rab enriched at primary cilia. These findings provide a basis for understanding how specific membrane trafficking pathways cooperate with the microtubule cytoskeleton to give rise to the primary cilia.

Specific Rab GTPase-activating proteins define the Shiga toxin and epidermal growth factor uptake pathways.

Rab family guanosine triphosphatases (GTPases) together with their regulators define specific pathways of membrane traffic within eukaryotic cells. In this study, we have investigated which Rab GTPase-activating proteins (GAPs) can interfere with the trafficking of Shiga toxin from the cell surface to the Golgi apparatus and studied transport of the epidermal growth factor (EGF) from the cell surface to endosomes. This screen identifies 6 (EVI5, RN-tre/USP6NL, TBC1D10A-C, and TBC1D17) of 39 predicted human Rab GAPs as specific regulators of Shiga toxin but not EGF uptake. We show that Rab43 is the target of RN-tre and is required for Shiga toxin uptake. In contrast, RabGAP-5, a Rab5 GAP, was unique among the GAPs tested and reduced the uptake of EGF but not Shiga toxin. These results suggest that Shiga toxin trafficking to the Golgi is a multistep process controlled by several Rab GAPs and their target Rabs and that this process is discrete from ligand-induced EGF receptor trafficking.

COP sets TRAPP for vesicles.

Intracellular transport vesicles identify their destination by a poorly understood process termed tethering. Recent work shows that in addition to its role in membrane-cargo selection, the COPII vesicle coat recruits TRAPPI, a cytosolic protein complex required for vesicle tethering.

A GTPase-activating protein controls Rab5 function in endocytic trafficking.

Rab-family GTPases are conserved regulators of membrane trafficking that cycle between inactive GDP-bound and activated GTP-bound states. A key determinant of Rab function is the lifetime of the GTP-bound state. As Rabs have a low intrinsic rate of GTP hydrolysis, this process is under the control of GTP-hydrolysis-activating proteins (GAPs). Due to the large number of Rabs and GAPs that are encoded by the human genome, it has proven difficult to assign specific functional relationships to these proteins. Here, we identify a Rab5-specific GAP (RabGAP-5), and show that RN-Tre (previously described as a Rab5 GAP) acts on Rab41. RabGAP-5 overexpression triggers a loss of the Rab5 effector EEA1 from endosomes and blocks endocytic trafficking. By contrast, depletion of RabGAP-5 results in increased endosome size, more endosome-associated EEA1, and disrupts the trafficking of EGF and LAMP1. RabGAP-5 therefore limits the amount of activated Rab5, and thereby regulates trafficking through endosomes.