A highly efficient transgene knock-in technology in clinically relevant cell types.

Inefficient knock-in of transgene cargos limits the potential of cell-based medicines. In this study, we used a CRISPR nuclease that targets a site within an exon of an essential gene and designed a cargo template so that correct knock-in would retain essential gene function while also integrating the transgene(s) of interest. Cells with non-productive insertions and deletions would undergo negative selection. This technology, called SLEEK (SeLection by Essential-gene Exon Knock-in), achieved knock-in efficiencies of more than 90% in clinically relevant cell types without impacting long-term viability or expansion. SLEEK knock-in rates in T cells are more efficient than state-of-the-art TRAC knock-in with AAV6 and surpass more than 90% efficiency even with non-viral DNA cargos. As a clinical application, natural killer cells generated from induced pluripotent stem cells containing SLEEK knock-in of CD16 and mbIL-15 show substantially improved tumor killing and persistence in vivo.

Autonomous and non-cell autonomous role of cilia in structural birth defects in mice.

Ciliopathies are associated with wide spectrum of structural birth defects (SBDs), indicating important roles for cilia in development. Here, we provide novel insights into the temporospatial requirement for cilia in SBDs arising from deficiency in Ift140, an intraflagellar transport (IFT) protein regulating ciliogenesis. Ift140-deficient mice exhibit cilia defects accompanied by wide spectrum of SBDs including macrostomia (craniofacial defects), exencephaly, body wall defects, tracheoesophageal fistula (TEF), randomized heart looping, congenital heart defects (CHDs), lung hypoplasia, renal anomalies, and polydactyly. Tamoxifen inducible CAGGCre-ER deletion of a floxed Ift140 allele between E5.5 to 9.5 revealed early requirement for Ift140 in left-right heart looping regulation, mid to late requirement for cardiac outflow septation and alignment, and late requirement for craniofacial development and body wall closure. Surprisingly, CHD were not observed with 4 Cre drivers targeting different lineages essential for heart development, but craniofacial defects and omphalocele were observed with Wnt1-Cre targeting neural crest and Tbx18-Cre targeting epicardial lineage and rostral sclerotome through which trunk neural crest cells migrate. These findings revealed cell autonomous role of cilia in cranial/trunk neural crest-mediated craniofacial and body wall closure defects, while non-cell autonomous multi-lineage interactions underlie CHD pathogenesis, revealing unexpected developmental complexity for CHD associated with ciliopathies.

Autonomous and non-cell autonomous etiology of ciliopathy associated structural birth defects.

Ciliopathies are associated with wide spectrum of structural birth defects (SBD), indicating important roles for cilia in development. Here we provide novel insights into the temporospatial requirement for cilia in SBDs arising from deficiency in Ift140 , an intraflagellar transport protein regulating ciliogenesis. Ift140 deficient mice exhibit cilia defects accompanied by wide spectrum of SBDs including macrostomia (craniofacial defects), exencephaly, body wall defects, tracheoesophageal fistula, randomized heart looping, congenital heart defects (CHD), lung hypoplasia, renal anomalies, and polydactyly. Tamoxifen inducible CAG-Cre deletion of a floxed Ift140 allele between E5.5 to 9.5 revealed early requirement for Ift140 in left-right heart looping regulation, mid to late requirement for cardiac outflow septation and alignment, and late requirement for craniofacial development and body wall closure. Surprisingly, CHD was not observed with four Cre drivers targeting different lineages essential for heart development, but craniofacial defects and omphalocele were observed with Wnt1-Cre targeting neural crest and Tbx18-Cre targeting epicardial lineage and rostral sclerotome through which trunk neural crest cells migrate. These findings revealed cell autonomous role of cilia in cranial/trunk neural crest mediated craniofacial and body wall closure defects, while non-cell autonomous multi-lineage interactions underlie CHD pathogenesis, revealing unexpected developmental complexity for CHD associated with ciliopathy.

Hypomorphic mutations of TRIP11 cause odontochondrodysplasia.

Odontochondrodysplasia (ODCD) is an unresolved genetic disorder of skeletal and dental development. Here, we show that ODCD is caused by hypomorphic TRIP11 mutations, and we identify ODCD as the nonlethal counterpart to achondrogenesis 1A (ACG1A), the known null phenotype in humans. TRIP11 encodes Golgi-associated microtubule-binding protein 210 (GMAP-210), an essential tether protein of the Golgi apparatus that physically interacts with intraflagellar transport 20 (IFT20), a component of the ciliary intraflagellar transport complex B. This association and extraskeletal disease manifestations in ODCD point to a cilium-dependent pathogenesis. However, our functional studies in patient-derived primary cells clearly support a Golgi-based disease mechanism. In spite of reduced abundance, residual GMAP variants maintain partial Golgi integrity, normal global protein secretion, and subcellular distribution of IFT20 in ODCD. These functions are lost when GMAP-210 is completely abrogated in ACG1A. However, a similar defect in chondrocyte maturation is observed in both disorders, which produces a cellular achondrogenesis phenotype of different severity, ensuing from aberrant glycan processing and impaired extracellular matrix proteoglycan secretion by the Golgi apparatus.

Implementation of Plate Imaging for Demonstration of Monoclonality in Biologics Manufacturing Development.

Monoclonality of mammalian cell lines used for production of biologics is a regulatory expectation and one of the attributes assessed as part of a larger process to ensure consistent quality of the biologic. Historically, monoclonality has been demonstrated through statistics generated from limiting dilution cloning or through verified flow cytometry methods. A variety of new technologies are now on the market with the potential to offer more efficient and robust approaches to generating and documenting a clonal cell line.Here we present an industry perspective on approaches for the application of imaging and integration of that information into a regulatory submission to support a monoclonality claim. These approaches represent the views of a consortium of companies within the BioPhorum Development Group and include case studies utilising imaging technology that apply scientifically sound approaches and efforts in demonstrating monoclonality. By highlighting both the utility of these alternative approaches and the advantages they bring over the traditional methods, as well as their adoption by industry leaders, we hope to encourage acceptance of their use within the biologics cell line development space and provide guidance for regulatory submission using these alternative approaches.LAY ABSTRACT: In the manufacture of biologics produced in mammalian cells, one recommendation by regulatory agencies to help ensure product consistency, safety, and efficacy is to produce the material from a monoclonal cell line derived from a single, progenitor cell. The process by which monoclonality is assured can be supplemented with single-well plate images of the progenitor cell. Here we highlight the utility of that imaging technology, describe approaches to verify the validity of those images, and discuss how to analyze that information to support a biologic filing application. This approach serves as an industry perspective to increased regulatory interest within the scope of monoclonality for mammalian cell culture-derived biologics.

Arf4 is required for Mammalian development but dispensable for ciliary assembly.

The primary cilium is a sensory organelle, defects in which cause a wide range of human diseases including retinal degeneration, polycystic kidney disease and birth defects. The sensory functions of cilia require specific receptors to be targeted to the ciliary subdomain of the plasma membrane. Arf4 has been proposed to sort cargo destined for the cilium at the Golgi complex and deemed a key regulator of ciliary protein trafficking. In this work, we show that Arf4 binds to the ciliary targeting sequence (CTS) of fibrocystin. Knockdown of Arf4 indicates that it is not absolutely required for trafficking of the fibrocystin CTS to cilia as steady-state CTS levels are unaffected. However, we did observe a delay in delivery of newly synthesized CTS from the Golgi complex to the cilium when Arf4 was reduced. Arf4 mutant mice are embryonic lethal and die at mid-gestation shortly after node formation. Nodal cilia appeared normal and functioned properly to break left-right symmetry in Arf4 mutant embryos. At this stage of development Arf4 expression is highest in the visceral endoderm but we did not detect cilia on these cells. In the visceral endoderm, the lack of Arf4 caused defects in cell structure and apical protein localization. This work suggests that while Arf4 is not required for ciliary assembly, it is important for the efficient transport of fibrocystin to cilia, and also plays critical roles in non-ciliary processes.

Analysis of ciliary membrane protein dynamics using SNAP technology.

The sensory functions of the primary cilium rely on receptors and other membrane proteins that are specifically sorted to the ciliary compartment, which is a subdomain of the plasma membrane. Defects in this process underlie a large number of human diseases, yet it is poorly understood. Thus, it is of great interest to understand the mechanisms by which the cell sorts and traffics proteins to the ciliary membrane. Here, we provide an overview of our method to study the sorting and trafficking of ciliary membrane proteins using SNAP technology. This technology enables pulse-chase analysis of the movement of proteins through the endomembrane system and onto the cilium.

IFT25 links the signal-dependent movement of Hedgehog components to intraflagellar transport.

The intraflagellar transport (IFT) system is required for building primary cilia, sensory organelles that cells use to respond to their environment. IFT particles are composed of about 20 proteins, and these proteins are highly conserved across ciliated species. IFT25, however, is absent from some ciliated organisms, suggesting that it may have a unique role distinct from ciliogenesis. Here, we generate an Ift25 null mouse and show that IFT25 is not required for ciliary assembly but is required for proper Hedgehog signaling, which in mammals occurs within cilia. Mutant mice die at birth with multiple phenotypes, indicative of Hedgehog signaling dysfunction. Cilia lacking IFT25 have defects in the signal-dependent transport of multiple Hedgehog components including Patched-1, Smoothened, and Gli2, and fail to activate the pathway upon stimulation. Thus, IFT function is not restricted to building cilia where signaling occurs, but also plays a separable role in signal transduction events.

The cytoplasmic tail of fibrocystin contains a ciliary targeting sequence.

Sensory functions of primary cilia rely on ciliary-localized membrane proteins, but little is known about how these receptors are targeted to the cilium. To further our understanding of this process, we dissected the ciliary targeting sequence (CTS) of fibrocystin, the human autosomal recessive polycystic kidney disease gene product. We show that the fibrocystin CTS is an 18-residue motif localized in the cytoplasmic tail. This motif is sufficient to target green fluorescent protein (GFP) to cilia of ciliated cells and targets GFP to lipid rafts if the cells are not ciliated. Rab8, but not several other Rabs implicated in ciliary assembly, binds to the CTS in a coimmunoprecipitation assay. Dominant-negative Rab8 interacts more strongly than wild-type or constitutively active Rab8, and coexpression of this dominant-negative mutant Rab8 blocks trafficking to the cilium. This suggests that the CTS functions by binding regulatory proteins like Rab8 to control trafficking through the endomembrane system and on to the cilium.

Characterization of mouse IFT complex B.

The primary cilium plays a key role in the development of mammals and in the maintenance of health. Primary cilia are assembled and maintained by the process of intraflagellar transport (IFT). In this work, we characterize mouse IFT complex B by identifying all of the mammalian orthologues of complex B and B-associated proteins previously identified in Chlamydomonas and Caenorhabditis and also identify a new component (IFT25/Hspb11) of complex B by database analysis. We tagged each of these proteins with the FLAG epitope and show that all except IFT172 and IFT20 localize to cilia and the peri-basal body or centrosomal region at the base of cilia. All of the proteins except IFT172 immunoprecipitate IFT88 indicating that they are co-assembled into a complex. IFT20 is the only complex B protein that localizes to the Golgi apparatus. However, overexpression of IFT54/Traf3ip1, the mouse orthologue of Dyf-11/Elipsa, displaces IFT20 from the Golgi apparatus. IFT54 does not localize to the Golgi complex nor does it interact with GMAP210, which is the protein that anchors IFT20 to the Golgi apparatus. This suggests that IFT54s effect on IFT20 is a dominant negative phenotype caused by its overexpression. Cell Motil. Cytoskeleton 2009. (c) 2009 Wiley-Liss, Inc.

Scanning electron microscopy to examine cells and organs.

Scanning electron microscopy is an excellent method for viewing the surface of cells and organs, and provides exquisite detail of surface projections. This method has a long history of use in the analysis of eukaryotic cilia and flagella. In this chapter, we provide methods used by our group to examine mouse kidneys and the embryonic node. The methods provided here can be used with little modification to examine other mammalian organs or used as a starting point to develop methods for use in other organisms.

The Golgin GMAP210/TRIP11 anchors IFT20 to the Golgi complex.

Eukaryotic cells often use proteins localized to the ciliary membrane to monitor the extracellular environment. The mechanism by which proteins are sorted, specifically to this subdomain of the plasma membrane, is almost completely unknown. Previously, we showed that the IFT20 subunit of the intraflagellar transport particle is localized to the Golgi complex, in addition to the cilium and centrosome, and hypothesized that the Golgi pool of IFT20 plays a role in sorting proteins to the ciliary membrane. Here, we show that IFT20 is anchored to the Golgi complex by the golgin protein GMAP210/Trip11. Mice lacking GMAP210 die at birth with a pleiotropic phenotype that includes growth restriction, ventricular septal defects of the heart, omphalocele, and lung hypoplasia. Cells lacking GMAP210 have normal Golgi structure, but IFT20 is no longer localized to this organelle. GMAP210 is not absolutely required for ciliary assembly, but cilia on GMAP210 mutant cells are shorter than normal and have reduced amounts of the membrane protein polycystin-2 localized to them. This work suggests that GMAP210 and IFT20 function together at the Golgi in the sorting or transport of proteins destined for the ciliary membrane.

Deletion of IFT20 in the mouse kidney causes misorientation of the mitotic spindle and cystic kidney disease.

Primary cilia project from the surface of most vertebrate cells and are thought to be sensory organelles. Defects in primary cilia lead to cystic kidney disease, although the ciliary mechanisms that promote and maintain normal renal function remain incompletely understood. In this work, we generated a floxed allele of the ciliary assembly gene Ift20. Deleting this gene specifically in kidney collecting duct cells prevents cilia formation and promotes rapid postnatal cystic expansion of the kidney. Dividing collecting duct cells in early stages of cyst formation fail to properly orient their mitotic spindles along the tubule, whereas nondividing cells improperly position their centrosomes. At later stages, cells lacking cilia have increased canonical Wnt signaling and increased rates of proliferation. Thus, IFT20 functions to couple extracellular events to cell proliferation and differentiation.

Functional analysis of an individual IFT protein: IFT46 is required for transport of outer dynein arms into flagella.

Intraflagellar transport (IFT), which is the bidirectional movement of particles within flagella, is required for flagellar assembly. IFT particles are composed of approximately 16 proteins, which are organized into complexes A and B. We have cloned Chlamydomonas reinhardtii and mouse IFT46, and show that IFT46 is a highly conserved complex B protein in both organisms. A C. reinhardtii insertional mutant null for IFT46 has short, paralyzed flagella lacking dynein arms and with central pair defects. The mutant has greatly reduced levels of most complex B proteins, indicating that IFT46 is necessary for complex B stability. A partial suppressor mutation restores flagellar length to the ift46 mutant. IFT46 is still absent, but levels of the other IFT particle proteins are largely restored, indicating that complex B is stabilized in the suppressed strain. Axonemal ultrastructure is restored, except that the outer arms are still missing, although outer arm subunits are present in the cytoplasm. Thus, IFT46 is specifically required for transporting outer arms into the flagellum.

The intraflagellar transport protein IFT20 is associated with the Golgi complex and is required for cilia assembly.

Eukaryotic cilia are assembled via intraflagellar transport (IFT) in which large protein particles are motored along ciliary microtubules. The IFT particles are composed of at least 17 polypeptides that are thought to contain binding sites for various cargos that need to be transported from their site of synthesis in the cell body to the site of assembly in the cilium. We show here that the IFT20 subunit of the particle is localized to the Golgi complex in addition to the basal body and cilia where all previous IFT particle proteins had been found. In living cells, fluorescently tagged IFT20 is highly dynamic and moves between the Golgi complex and the cilium as well as along ciliary microtubules. Strong knock down of IFT20 in mammalian cells blocks ciliary assembly but does not affect Golgi structure. Moderate knockdown does not block cilia assembly but reduces the amount of polycystin-2 that is localized to the cilia. This work suggests that IFT20 functions in the delivery of ciliary membrane proteins from the Golgi complex to the cilium.