Landing Pad Research Articles

CHOK1SV GS-KO SSI expression system: A combination of the Fer1L4 locus and glutamine synthetase selection.

There are an ever-increasing number of biopharmaceutical candidates in clinical trials fueling an urgent need to streamline the cell line development process. A critical part of the process is the methodology used to generate and screen candidate cell lines compatible with GMP manufacturing processes. The relatively large amount of clone phenotypic variation observed from conventional "random integration" (RI)-based cell line construction is thought to be the result of a combination of the position variegation effect, genome plasticity and clonal variation. Site-specific integration (SSI) has been used by several groups to temper the influence of the position variegation effect and thus reduce variability in expression of biopharmaceutical candidates. Following on from our previous reports on the application of the Fer1L4 locus for SSI in CHOK1SV (10E9), we have combined this locus and a CHOK1SV glutamine synthetase knockout (GS-KO) host to create an improved expression system. The host, CHOK1SV GS-KO SSI (HD7876), was created by homology directed integration of a targetable landing pad flanked with incompatible Frt sequences in the Fer1L4 gene. The targeting vector contains a promoterless GS expression cassette and monoclonal antibody (mAb) expression cassettes, flanked by Frt sites compatible with equivalent sites flanking the landing pad in the host cell line. SSI clones expressing four antibody candidates, selected in a streamlined cell line development process, have mAb titers which rival RI (1.0-4.5g/L) and robust expression stability (100% of clones stable through the 50 generation "manufacturing window" which supports commercial manufacturing at 12,000 L bioreactor scale).

Towards the Development of an Automatic UAV-Based Indoor Environmental Monitoring System: Distributed Off-Board Control System for a Micro Aerial Vehicle

This research aims to develop an automatic unmanned aerial vehicle (UAV)-based indoor environmental monitoring system for the acquisition of data at a very fine scale to detect rapid changes in environmental features of plants growing in greenhouses. Due to the complexity of the proposed research, in this paper we proposed an off-board distributed control system based on visual input for a micro aerial vehicle (MAV) able to hover, navigate, and fly to a desired target location without considerably affecting the effective flight time. Based on the experimental results, the MAV was able to land on the desired location within a radius of about 10 cm from the center point of the landing pad, with a reduction in the effective flight time of about 28%.

SLCO1B1: Application and Limitations of Deep Mutational Scanning for Genomic Missense Variant Function.

SLCO1B1 (solute carrier organic anion transporter family member 1B1) is an important transmembrane hepatic uptake transporter. Genetic variants in the SLCO1B1 gene have been associated with altered protein folding, resulting in protein degradation and decreased transporter activity. Next-generation sequencing (NGS) of pharmacogenes is being applied increasingly to associate variation in drug response with genetic sequence variants. However, it is difficult to link variants of unknown significance with functional phenotypes using “one-at-a-time” functional systems. Deep mutational scanning (DMS) using a “landing pad cell–based system” is a high-throughput technique designed to analyze hundreds of gene open reading frame (ORF) missense variants in a parallel and scalable fashion. We have applied DMS to analyze 137 missense variants in the SLCO1B1 ORF obtained from the Exome Aggregation Consortium project. ORFs containing these variants were fused to green fluorescent protein and were integrated into “landing pad” cells. Florescence-activated cell sorting was performed to separate the cells into four groups based on fluorescence readout indicating protein expression at the single cell level. NGS was then performed and SLCO1B1 variant frequencies were used to determine protein abundance. We found that six variants not previously characterized functionally displayed less than 25% and another 12 displayed approximately 50% of wild-type protein expression. These results were then functionally validated by transporter studies. Severely damaging variants identified by DMS may have clinical relevance for SLCO1B1-dependent drug transport, but we need to exercise caution since the relatively small number of severely damaging variants identified raise questions with regard to the application of DMS to intrinsic membrane proteins such as organic anion transporter protein 1B1.Significance StatementThe functional implications of a large numbers of open reading frame (ORF) “variants of unknown significance” (VUS) in transporter genes have not been characterized. This study applied deep mutational scanning to determine the functional effects of VUS that have been observed in the ORF of SLCO1B1(solute carrier organic anion transporter family member 1B1). Several severely damaging variants were identified, studied, and validated. These observations have implications for both the application of deep mutational scanning to intrinsic membrane proteins and for the clinical effect of drugs and endogenous compounds transported by SLCO1B1.

A versatile platform for locus-scale genome rewriting and verification

Routine rewriting of loci associated with human traits and diseases would facilitate their functional analysis. However, existing DNA integration approaches are limited in terms of scalability and portability across genomic loci and cellular contexts. We describe Big-IN, a versatile platform for targeted integration of large DNAs into mammalian cells. CRISPR/Cas9-mediated targeting of a landing pad enables subsequent recombinase-mediated delivery of variant payloads and efficient positive/negative selection for correct clones in mammalian stem cells. We demonstrate integration of constructs up to 143 kb, and an approach for one-step scarless delivery. We developed a staged pipeline combining PCR genotyping and targeted capture sequencing for economical and comprehensive verification of engineered stem cells. Our approach should enable combinatorial interrogation of genomic functional elements and systematic locus-scale analysis of genome function.

Development of recombinase-based targeted integration systems for production of exogenous proteins using transposon-mediated landing pads

Abstract Current methods for stably expressing recombinant protein therapeutics in CHO cells often rely on random or semi-random genomic integration events which result in a widely heterogeneous cell population. Consequently, a significant portion of cell line development efforts involves extensive pool and clone screening to identify clones with high expression, growth, and product quality. In this study, we developed two targeted integration systems that express high levels of recombinant protein in CHO cells. We first generated two clonal cell lines stably expressing enhanced green fluorescent protein (eGFP) reporter landing pads in genomic hot spots. We then demonstrated functional integration of several donor vectors encoding both monoclonal antibodies and a Fc-fusion molecule using Cre or PhiC31 recombinase mediated integration. TLA and PCR characterization of integrated cell lines showed correct targeting of landing pads. Post-integration enrichment for fully saturated landing pads using GCV increased recombinant protein titer by 2–2.5-fold and specific productivity by ∼3.4-fold with observed potential off-target random integration detected only following GCV enrichment. By targeting predefined genomic locations that are known to support high expression of an exogenous protein, several cell lines expressing different recombinant protein therapeutics can easily be established with a high degree of specificity and reproducibility.

Genetic rearrangement during site specific integration event facilitates cell line development of a bispecific molecule.

Site specific integration (SSI) expression systems offer robust means of generating highly productive and stable cell lines for traditional monoclonal antibodies. As complex modalities such as antibody‐like molecules comprised of greater than two peptides become more prevalent, greater emphasis needs to be placed on the ability to produce appreciable quantities of the correct product of interest (POI). The ability to screen several transcript stoichiometries could play a large role in ensuring high amounts of the correct POI. Here we illustrate implementation of an SSI expression system with a single site of integration for development and production of a multi‐chain, bi‐specific molecule. A SSI vector with a single copy of all of the genes of interest was initially selected for stable Chinese hamster ovary transfection. While the resulting transfection pools generated low levels of the desired heterodimer, utilizing an intensive clone screen strategy, we were able to identify clones having significantly higher levels of POI. In‐depth genotypic characterization of clones having the desirable phenotype revealed that a duplication of the light chain within the landing pad was responsible for producing the intended molecule. Retrospective transfection pool analysis using a vector configuration mimicking the transgene configuration found in the clones, as well as other vector configurations, yielded more favorable results with respect to % POI. Overall, the study demonstrated that despite the theoretical static nature of the SSI expression system, enough heterogeneity existed to yield clones having significantly different transgene phenotypes/genotypes and support production of a complex multi‐chain molecule.

An Optimized Genome-Wide Virus-Free CRISPR Screen for Mammalian Cells

Pooled CRISPR screens have been widely applied to mammalian and other organisms to elucidate the interplay between genes and phenotypes of interest. The most popular method for delivering the CRISPR components into mammalian cells is lentivirus based. However, lentivirus is not always an option, virus-free (VF) protocols are starting to emerge. Here we demonstrate an improved virus-free (VF) genome-wide CRISPR screening platform for Chinese hamster ovary (CHO) cells with 75,488 gRNAs targeting 15,028 genes. Each gRNA expression cassette in the library is precisely integrated into a genomic landing pad, resulting in a very high percentage of single gRNA insertion and minimal clonal variation. Using this platform, we perform a negative selection screen on cell proliferation that identifies 1980 genes that affect proliferation in CHO and a positive selection screen on toxic endoplasmic reticulum (ER) stress inducer, tunicamycin, that identifies 77 gene KOs that improve survivability.

Sacrificial Volume Materials for Small Hole Generation in Low-Temperature Cofired Ceramics

The creation of hole, cavity or channel structures in low-temperature cofired ceramics (LTCCs), using different sacrificial volume materials (SVMs) was tested. The main functionality of the SVMs should be: easy application into the holes; protection of holes during lamination; uninhibited lamination between layers; and, during firing should burn out before the pores of the LTCCs close, to leave the empty holes clear of any residue. Five different materials were tested—hydroxyethyl cellulose (HEC) 2 wt%, HEC 5 wt%, agar-agar, gelatin, and commercial carbon paste—and compared to a reference sample where no SVMs were used. In all cases, lamination parameters were minimised in order to preserve the tested hole structures. Matrixes with holes ranging from 158 to 268 µm, with pitches of 573 µm in a green state, were tested. The agar-agar caused ceramic deformation as a result of thermal burst during firing and the lamination between the layers was compromised. The carbon paste was difficult to handle, requiring extra equipment for hole filling and incomplete filling of the larger holes. Traces of carbon paste were left as a landing pad on top of the holes, inhibiting lamination at these areas. The gelatin and the 2 wt% and 5 wt% hydroxyethylcellulose (HEC 2 wt% and HEC 5 wt%) filled all holes completely, and also worked as adhesive-assisted lamination (AAL) materials with excellent lamination between layers. Excellent lamination was also observed in the no-SVM case. Thermogravimetric analysis (TGA) and scanning electron microscopy (SEM) with energy-dispersive X-ray (EDX) analyses established that, for all SVMs tested, the remaining residue is negligible after firing. As a result, the HEC 2 wt% material was considered ideal for use as an SVM.

Charging Platform of Chess-Pad Configuration for Unmanned Aerial Vehicle (UAV)

The authors of this study designed and optimized a charging landing pad system that mitigates the landing accuracy issues of unmanned aerial vehicles (UAVs). The study looks at the charging process, energy conversion during periodic landing on a unique platform, and an onboard and on-ground scheme design procedure. The circuit is fixed on the UAV platform and comprises six integrated bridge rectifier diodes to alter the four connection pin terminals’ charge polarity. The inclusion of a current indicator shows the flow of charge during successful docking. The charging platform consists of square conductive copper plates of specific dimensions that provide positive and negative polarity in a chess form to ensure the contact of various polarities. This design considers two power supply options: a solar panel and a standard mains supply. The contact point coordinate probability when landing is the crucial aspect of this design. A first version of the proposed system was implemented to measure its effectiveness for commercial drones. This system provides an automated recharge station with reliable performance. Numerical experiments showed that the system’s energy conversion remains efficient regardless of drone orientation over the platform or the environment’s nature.

Bacterial genome editing by coupling Cre-lox and CRISPR-Cas9 systems.

The past decade has been a golden age for microbiology, marked by the discovery of an unprecedented increase in the number of novel bacterial species. Yet gaining biological knowledge of those organisms has not kept pace with sequencing efforts. To unlock this genetic potential there is an urgent need for generic (i.e. non-species specific) genetic toolboxes. Recently, we developed a method, termed chassis-independent recombinase-assisted genome engineering (CRAGE), enabling the integration and expression of large complex gene clusters directly into the chromosomes of diverse bacteria. Here we expand upon this technology by incorporating CRISPR-Cas9 allowing precise genome editing across multiple bacterial species. To do that we have developed a landing pad that carries one wild-type and two mutant lox sites to allow integration of foreign DNA at two locations through Cre-lox recombinase-mediated cassette exchange (RMCE). The first RMCE event is to integrate the Cas9 and the DNA repair protein genes RecET, and the second RMCE event enables the integration of customized sgRNA and a repair template. Following this workflow, we achieved precise genome editing in four different gammaproteobacterial species. We also show that the inserted landing pad and the entire editing machinery can be removed scarlessly after editing. We report here the construction of a single landing pad transposon and demonstrate its functionality across multiple species. The modular design of the landing pad and accessory vectors allows design and assembly of genome editing platforms for other organisms in a similar way. We believe this approach will greatly expand the list of bacteria amenable to genetic manipulation and provides the means to advance our understanding of the microbial world.

Multicopy Targeted Integration for Accelerated Development of High-Producing Chinese Hamster Ovary Cells.

The ever-growing biopharmaceutical industry relies on the production of recombinant therapeutic proteins in Chinese hamster ovary (CHO) cells. The traditional timelines of CHO cell line development can be significantly shortened by the use of targeted gene integration (TI). However, broad use of TI has been limited due to the low specific productivity (qP) of TI-generated clones. Here, we show a 10-fold increase in the qP of therapeutic glycoproteins in CHO cells through the development and optimization of a multicopy TI method. We used a recombinase-mediated cassette exchange (RMCE) platform to investigate the effect of gene copy number, 5' and 3' gene regulatory elements, and landing pad features on qP. We evaluated the limitations of multicopy expression from a single genomic site as well as multiple genomic sites and found that a transcriptional bottleneck can appear with an increase in gene dosage. We created a dual-RMCE system for simultaneous multicopy TI in two genomic sites and generated isogenic high-producing clones with qP of 12-14 pg/cell/day and product titer close to 1 g/L in fed-batch. Our study provides an extensive characterization of the multicopy TI method and elucidates the relationship between gene copy number and protein expression in mammalian cells. Moreover, it demonstrates that TI-generated CHO cells are capable of producing therapeutic proteins at levels that can support their industrial manufacture.

Precision design of stable genetic circuits carried in highly‐insulated E. coli genomic landing pads

Genetic circuits have many applications, from guiding living therapeutics to ordering process in a bioreactor, but to be useful they have to be genetically stable and not hinder the host. Encoding circuits in the genome reduces burden, but this decreases performance and can interfere with native transcription. We have designed genomic landing pads in Escherichia coli at high‐expression sites, flanked by ultrastrong double terminators. DNA payloads >8 kb are targeted to the landing pads using phage integrases. One landing pad is dedicated to carrying a sensor array, and two are used to carry genetic circuits. NOT/NOR gates based on repressors are optimized for the genome and characterized in the landing pads. These data are used, in conjunction with design automation software (Cello 2.0), to design circuits that perform quantitatively as predicted. These circuits require fourfold less RNA polymerase than when carried on a plasmid and are stable for weeks in a recA+ strain without selection. This approach enables the design of synthetic regulatory networks to guide cells in environments or for applications where plasmid use is infeasible.

CRISPR-addressable yeast strains with applications in human G protein–coupled receptor profiling and synthetic biology

Genome stability is essential for engineering cell-based devices and reporter systems. With the advent of CRISPR technology, it is now possible to build such systems by installing the necessary genetic parts directly into an organism's genome. Here, we used this approach to build a set of 10 versatile yeast-based reporter strains for studying human G protein-coupled receptors (GPCRs), the largest class of membrane receptors in humans. These reporter strains contain the necessary genetically encoded parts for studying human GPCR signaling in yeast, as well as four CRISPR-addressable expression cassettes, i.e. landing pads, installed at known safe-harbor sites in the yeast genome. We showcase the utility of these strains in two applications. First, we demonstrate that increasing GPCR expression by incrementally increasing GPCR gene copy number potentiates Gα coupling of the pharmacologically dark receptor GPR68. Second, we used two CRISPR-addressable landing pads for autocrine activation of a GPCR (the somatostatin receptor SSTR5) with its peptide agonist SRIF-14. The utility of these reporter strains can be extended far beyond these select examples to include applications such as nanobody development, mutational analysis, drug discovery, and studies of GPCR chaperoning. Additionally, we present a BY4741 yeast strain created for broad applications in the yeast and synthetic biology communities that contains only the four CRISPR-addressable landing pads. The general utility of these yeast strains provides an inexpensive, scalable, and easy means of installing and expressing genes directly from the yeast genome to build genome-barcoded sensors, reporter systems, and cell-based factories.

Comprehensive Analysis of Genomic Safe Harbors as Target Sites for Stable Expression of the Heterologous Gene in HEK293 Cells.

Human cell lines are being increasingly used as host cells to produce therapeutic glycoproteins, due to their human glycosylation machinery. In an attempt to develop a platform for generating isogenic human cell lines producing therapeutic proteins based on targeted integration, three well-known human genomic safe harbors (GSHs)-AAVS1, CCR5, and human ROSA26 loci-were evaluated with respect to the transgene expression level and stability in human embryonic kidney (HEK293) cells. Among the three GSHs, the AAVS1 locus showed the highest eGFP expression with the highest homogeneity. Transgene expression at the AAVS1 locus was sustained without selection for approximately 3 months. Furthermore, the CMV promoter showed the highest expression, followed by the EF1α, SV40, and TK promoters at the AAVS1 locus. Master cell lines were created using CRISPR/Cas9-mediated integration of the landing pad into the AAVS1 locus and were used for faster generation of recombinant cell lines that produce therapeutic proteins with recombinase-mediated cassette exchange.

The Principles and Organization of Air Traffic in Military Operations: Experiences from the Mission in Iraq

This article analyzes the principles and organization of the operation of the military runways used by Polish and Allied aircrafts during missions in Iraq. The main goal of the article is to describe the organization and method of air traffic management by the Polish air traffic controllers (ATC). It depicts the method of planning and preparing aviation operations, the principles of air traffic organization, which were the responsibility of the Polish controllers, and the rules for the traffic above the airstrip. Among the particular problems that are discussed, there are the rules of airspace segmentation, the introduced solutions for air traffic control, and the use of airport infrastructure. The experiences discussed include valuable information regarding the process of securing air traffic that can be helpful in the organization of landing pads and in preparation of both Polish and allied military contingent aviation personnel for future aboard operations. This paper presents the results of the research carried out with the ATC personnel performing tasks in Iraq.

Complex Trait Loci in Maize Enabled by CRISPR-Cas9 Mediated Gene Insertion.

Modern maize hybrids often contain biotech and native traits. To-date all biotech traits have been randomly inserted in the genome. Consequently, developing hybrids with multiple traits is expensive, time-consuming, and complex. Here we report using CRISPR-Cas9 to generate a complex trait locus (CTL) to facilitate trait stacking. A CTL consists of multiple preselected sites positioned within a small well-characterized chromosomal region where trait genes are inserted. We generated individual lines, each carrying a site-specific insertion landing pad (SSILP) that was targeted to a preselected site and capable of efficiently receiving a transgene via recombinase-mediated cassette exchange. The selected sites supported consistent transgene expression and the SSILP insertion had no effect on grain yield. We demonstrated that two traits residing at different sites within a CTL can be combined via genetic recombination. CTL technology is a major step forward in the development of multi-trait maize hybrids.

Formation of appearance of helicopter-type unmanned airvehicle with consideration of landing on complex-moving platform

The article considers an approach to the formation of the image of an unmanned aircraft, taking into account the landing on a complex-moving platform. The main parameters characterizing the appearance of a helicopter-type unmanned air vehicle are analyzed and the task of appearance forming is formulated. Two necessary conditions for landing on a ship, such as static and dynamic compatibility are defined and formulated. The formation of the technical appearance of the ship unmanned helicopter is based on the interrelated processes of determination of the following parameters: volume-mass, structural-power, aerodynamic, power plant and take-off and landing devices. The formulation of the initial parameters of the landing pad movement on the ship is considered and the main dependencies of pitching are formulated. On the basis of the conducted researches and taking into account the accepted restrictions, the authors propose technical ways to solve the problem.

Recombinase‐mediated Method for Multi‐scale Genomic Rearrangements

With the widespread use of genomic editing tools like CRISPR/Cas9, scientists have gained the ability to modify a host genome with high precision. Although these technological advancements have improved throughout the years, there is still a need for better methods to build more sophisticated multi‐part biological circuits. For this reason, we have employed the use of a genomic integration technique, landing pads, which utilized site‐specific recombinases to insert relatively large DNA assemblies into a well‐defined location the host genome. Using the CRISPR/Cas9 system, we knocked‐in an 8kb landing pad repair template into the AAVSl safe harbor locus of HEK293T cells. After single‐cell sorting and verifying the genotype of our clonal cell line, we used the BxB1 recombinase to integrate a destination plasmid into the landing pad site. Using microscopy, flow cytometry, and PCR genotyping, we demonstrate almost complete replacement of the original landing pad template with our destination vector species within 12 days. Here we present an approach that enables the rapid creation of mammalian cell lines harboring complex, multi‐part regulatory circuits at single copy. This platform allows for precise and tunable control of gene expression profiles, providing an excellent opportunity to engineer safer and more sophisticated cell‐‐based therapies.Support or Funding InformationNational Institute of General Medical Sciences of the National Institutes of Health under linked Award Numbers SC1GM111172, RL5GM118969, TL4GM118971, UL1GM118970, and 5G12MD007592.

CYP2C9 and CYP2C19: Deep Mutational Scanning and Functional Characterization of Genomic Missense Variants.

Single nucleotide variants in the open reading frames (ORFs) of pharmacogenes are important causes of interindividual variability in drug response. The functional characterization of variants of unknown significance within ORFs remains a major challenge for pharmacogenomics. Deep mutational scanning (DMS) is a high‐throughput technique that makes it possible to analyze the functional effect of hundreds of variants in a parallel and scalable fashion. We adapted a “landing pad” DMS system to study the function of missense variants in the ORFs of cytochrome P450 family 2 subfamily C member 9 (CYP2C9) and cytochrome P450 family 2 subfamily C member 19 (CYP2C19). We studied 230 observed missense variants in the CYP2C9 and CYP2C19 ORFs and found that 19 of 109 CYP2C9 and 36 of 121 CYP2C19 variants displayed less than ~ 25% of the wild‐type protein expression, a level that may have clinical relevance. Our results support DMS as an efficient method for the identification of damaging ORF variants that might have potential clinical pharmacogenomic application.

Mammalian RNA Decay Pathways Are Highly Specialized and Widely Linked to Translation.

SummaryRNA decay is crucial for mRNA turnover and surveillance and misregulated in many diseases. This complex system is challenging to study, particularly in mammals, where it remains unclear whether decay pathways perform specialized versus redundant roles. Cytoplasmic pathways and links to translation are particularly enigmatic. By directly profiling decay factor targets and normal versus aberrant translation in mouse embryonic stem cells (mESCs), we uncovered extensive decay pathway specialization and crosstalk with translation. XRN1 (5′-3′) mediates cytoplasmic bulk mRNA turnover whereas SKIV2L (3′-5′) is universally recruited by ribosomes, tackling aberrant translation and sometimes modulating mRNA abundance. Further exploring translation surveillance revealed AVEN and FOCAD as SKIV2L interactors. AVEN prevents ribosome stalls at structured regions, which otherwise require SKIV2L for clearance. This pathway is crucial for histone translation, upstream open reading frame (uORF) regulation, and counteracting ribosome arrest on small ORFs. In summary, we uncovered key targets, components, and functions of mammalian RNA decay pathways and extensive coupling to translation.