Genetic Engineering Of Cells Research Articles

Effects of process intensification on homogeneity of an IgG1:κ monoclonal antibody during perfusion culture

The pharmaceutical industry employs various strategies to improve cell productivity. These strategies include process intensification, culture media improvement, clonal selection, media supplementation and genetic engineering of cells. However, improved cell productivity has inherent risk of impacting product quality attributes (PQA). PQAs may affect the products’ efficacy via stability, bioavailability, or in vivo bioactivity. Variations in manufacturing process may introduce heterogeneity in the products by altering the type and extent of N-glycosylation, which is a PQA of therapeutic proteins. We investigated the effect of different cell densities representing increasing process intensification in a perfusion cell culture on the production of an IgG1-κ monoclonal antibody from a CHO-K1 cell line. This antibody is glycosylated both on light chain and heavy chain. Our results showed that the contents of glycosylation of IgG1-κ mAb increased in G0F and fucosylated type glycans as a group, whereas sialylated type glycans decreased, for the mAb whole protein. Overall, significant differences were observed in amounts of G0F, G1F, G0, G2FS1, and G2FS2 type glycans across all process intensification levels. G2FS2 and G2 type N-glycans were predominantly quantifiable from light chain rather than heavy chain. It may be concluded that there is a potential impact to product quality attributes of therapeutic proteins during process intensification via perfusion cell culture that needs to be assessed. Since during perfusion cell culture the product is collected throughout the duration of the process, lot allocation needs careful attention to process parameters, as PQAs are affected by the critical process parameters (CPPs).Key points• Molecular integrity may suffer with increasing process intensity.• Galactosylated and sialylated N-glycans may decrease.• Perfusion culture appears to maintain protein charge structure.

Sustained transgene expression from sleeping beauty DNA transposons containing a core fragment of the HNRPA2B1-CBX3 ubiquitous chromatin opening element (UCOE)

BackgroundDNA transposon-based vectors are effective nonviral tools for gene therapy and genetic engineering of cells. However, promoter DNA methylation and a near-random integration profile, which can result in transgene integration into heterochromatin, renders such vectors vulnerable to transcriptional repression. Therefore, to secure persistent transgene expression it may be necessary to protect transposon-embedded transgenes with anti-transcriptional silencing elements.ResultsWe compare four different protective strategies in CHO-K1 cells. Our findings show robust protection from silencing of transgene cassettes mediated by the ubiquitous chromatin-opening element (UCOE) derived from the HNRPA2B1-CBX3 locus. Using a bioinformatic approach, we define a shorter HNRPA2B1-CBX3 UCOE core fragment and demonstrate that this can robustly maintain transgene expression after extended passaging of CHO-K1 cells carrying DNA transposon vectors equipped with this protective feature.ConclusionsOur findings contribute to the understanding of the mechanism of HNRPA2B1-CBX3 UCOE-based transgene protection and support the use of a correctly oriented core fragment of this UCOE for DNA transposon vector-based production of recombinant proteins in CHO-K1 cells.

Direct One-Step Fluorescent Labeling of O-GlcNAc-Modified Proteins in Live Cells Using Metabolic Intermediates.

The modification of proteins with O-linked N-acetylglucosamine ( O-GlcNAc) by the enzyme O-GlcNAc transferase (OGT) has emerged as an important regulator of cellular physiology. Metabolic labeling strategies to monitor O-GlcNAcylation in cells have proven of great value for uncovering the molecular roles of O-GlcNAc. These strategies rely on two-step labeling procedures, which limits the scope of experiments that can be performed. Here, we report on the creation of fluorescent uridine 5'-diphospho- N-acetylglucosamine (UDP-GlcNAc) analogues in which the N-acyl group of glucosamine is modified with a suitable linker and fluorophore. Using human OGT, we show these donor sugar substrates permit direct monitoring of OGT activity on protein substrates in vitro. We show that feeding cells with a corresponding fluorescent metabolic precursor for the last step of the hexosamine biosynthetic pathway (HBP) leads to its metabolic assimilation and labeling of O-GlcNAcylated proteins within live cells. This one-step metabolic feeding strategy permits labeling of O-GlcNAcylated proteins with a fluorescent glucosamine-nitrobenzoxadiazole (GlcN-NBD) conjugate that accumulates in a time- and dose-dependent manner. Because no genetic engineering of cells is required, we anticipate this strategy should be generally amenable to studying the roles of O-GlcNAc in cellular physiology as well as to gain an improved understanding of the regulation of OGT within cells. The further expansion of this one-step in-cell labeling strategy should enable performing a range of experiments including two-color pulse chase experiments and monitoring OGT activity on specific protein substrates in live cells.

A review on heavy metals biosorption in the environment

Heavy metal refers to any metallic chemical element that has a relatively high density and is toxic or poisonous at low concentrations. Examples of heavy metals include mercury (Hg), cadmium (Cd), arsenic (As), chromium (Cr), thallium (Tl) and lead (Pb). Little amounts of some heavy metals are needed by living organisms, however excessive levels of these metals can be harmful to the organisms due to their level of toxicity and accumulation behaviour. Different methods such as electrodeposition, electrocoagulation and nanofiltration system have been used to decontaminate the environment from adverse effect of these pollutants yet most of the methods used are ineffective. Biosorption is the removal of organic and inorganic substances from solution by biological material. Cheap biosorbents for the removal of metals are bacteria, fungi, algae, plants, industrial wastes and agricultural wastes. There are many mechanisms involved in biosorption some of which are not fully understood, examples are precipitation, ion exchange, complexation and adsorption. The efficiency of biosorption depends on many factors such as, temperature, characteristics of the biomass, pH, surface area to volume ratio, metal affinity to the biosorbent, concentration and characteristics of the biomass. Compared to other methods biosorption is operated over a wide range of physiochemical conditions and it uses naturally rich renewable biomaterials that can be cheaply produced. However, the potential for biological process improvement (for example through genetic engineering of cells) is restricted because cells are not metabolizing. Biosorption is in its developmental stages and further improvement in both performance and costs can be expected in future.

Lentiviral Vectors for the Engineering of Implantable Cells Secreting Recombinant Antibodies.

The implantation of genetically modified cells is considered for the chronic delivery of therapeutic recombinant proteins in vivo. In the context of gene therapy, the genetic engineering of cells faces two main challenges. First, it is critical to generate expandable cell sources, which can maintain stable high productivity of the recombinant protein of interest over time, both in culture and after transplantation. In addition, gene transfer techniques need to be developed to engineer cells synthetizing complex polypeptides, such as recombinant monoclonal antibodies, to broaden the range of potential therapeutic applications. Here, we provide a workflow for the use of lentiviral vectors as a flexible tool to generate antibody-producing cells. In particular, lentiviral vectors can be used to genetically engineer the cell types compatible with encapsulation devices protecting the implanted cells from the host immune system. Detailed methods are provided for the design and production of lentiviral vectors, optimization of cell transduction, as well as for the quantification and quality control of the produced recombinant antibody.

Programming languages for circuit design.

This chapter provides an overview of a programming language for Genetic Engineering of Cells (GEC). A GEC program specifies a genetic circuit at a high level of abstraction through constraints on otherwise unspecified DNA parts. The GEC compiler then selects parts which satisfy the constraints from a given parts database. GEC further provides more conventional programming language constructs for abstraction, e.g., through modularity. The GEC language and compiler is available through a Web tool which also provides functionality, e.g., for simulation of designed circuits.

Field-deployable whole-cell bioluminescent biosensors: so near and yet so far

The use of smart supports and bioinspired materials to confine living cells and use them for field-deployable biosensors has recently attracted much attention. In particular, bioluminescent whole-cell biosensors designed to respond to different analytes or classes of analyte have been successfully implemented in portable and cost-effective analytical devices. Significant advances in detection technology, biomaterial science, and genetic engineering of cells have recently been reported. Now the challenge is to move from benchtop traditional cell-based assays to portable biosensing devices. Improvement of the analytical performance of these biosensors depends on the availability of optimized bioluminescent reporters, and promising approaches that go beyond reporter gene technology are emerging. To enable handling of cells as ready-to-use reagents, nature-inspired strategies have been used, with the objective of keeping cells in a dormant state until use. Several issues must still be investigated, for example long-term viability of cells, the possibility of performing real-time analysis, and multiplexing capability.

Meeting report: The Cambridge BioDesign TechEvent – Synthetic Biology, a new “Age of Wonder”?

Meeting report: The Cambridge BioDesign TechEvent – Synthetic Biology, a new “Age of Wonder”?

Towards the rational design of synthetic cells with prescribed population dynamics

The rational design of synthetic cell populations with prescribed behaviours is a long-standing goal of synthetic biology, with the potential to greatly accelerate the development of biotechnological applications in areas ranging from medical research to energy production. Achieving this goal requires well-characterized components, modular implementation strategies, simulation across temporal and spatial scales and automatic compilation of high-level designs to low-level genetic parts that function reliably inside cells. Many of these steps are incomplete or only partially understood, and methods for integrating them within a common design framework have yet to be developed. Here, we address these challenges by developing a prototype framework for designing synthetic cells with prescribed population dynamics. We extend the genetic engineering of cells (GEC) language, originally developed for programming intracellular dynamics, with cell population factors such as cell growth, division and dormancy, together with spatio-temporal simulation methods. As a case study, we use our framework to design synthetic cells with predator–prey interactions that, when simulated, produce complex spatio-temporal behaviours such as travelling waves and spatio-temporal chaos. An analysis of our design reveals that environmental factors such as density-dependent dormancy and reduced extracellular space destabilize the population dynamics and increase the range of genetic variants for which complex spatio-temporal behaviours are possible. Our findings highlight the importance of considering such factors during the design process. We then use our analysis of population dynamics to inform the selection of genetic parts, which could be used to obtain the desired spatio-temporal behaviours. By identifying, integrating and automating key stages of the design process, we provide a computational framework for designing synthetic systems, which could be tested in future laboratory studies.

Adoptive Cell Transfer Therapy

Adoptive cell transfer therapy has developed into a potent and effective treatment for patients with metastatic melanoma. Current application of this therapy relies on the ex vivo generation of highly active, highly avid tumor-reactive lymphocyte cultures from endogenous tumor infiltrating lymphocytes or on the genetic engineering of cells using antigen receptor genes to express de novo tumor antigen recognition. When autologous anti-tumor lymphocyte cultures are administered to patients with high-dose interleukin (IL)-2 following a lymphodepleting conditioning regimen, the cells can expand in vivo, traffic to tumor, and mediate tumor regression and durable objective clinical responses. Current investigation seeks to improve the methods for generating and administering the lymphocyte cultures, and future clinical trials aim to improve durable response rates and extend the patient populations that are candidates for treatment.

ELECTROPORATION: BIO-ELECTROCHEMICAL MASS TRANSFER AT THE NANO SCALE

This article provides a brief review of the field of electroporation and introduces a new microdevice that facilitates studies to test theories, gain understanding, and control this important biomedical technology. Electroporation, a bio-electrochemical process whose fundamentals are not yet understood, is a means of permeating the cell membrane by applying a voltage across the cell and forming nano-scale pores in the membrane. It has become an important field in biotechnology and medicine for the controlled introduction of macromolecules, such as gene constructs and drugs, into various cells. It is viewed as an engineering alternative to biological techniques for the genetic engineering of cells. To study and control electroporation, we have created a low-cost microelectroporation chip that incorporates a live biological cell with an electric circuit. The device revealed an important behavior of cells in electrical fields. They produce measurable electrical information about the electroporation state of the cell that may enable precise control of the process. The device can be used to facilitate fundamental studies of electroporation and can become useful in providing precise control over biotechnological processes.

Immune reconstitution in AIDS: oncologic implications and hematologic approaches.

Combination anti-retroviral therapy for HIV disease has profoundly altered the nature of the AIDS epidemic. Mitigating the impact of an uncontrollable decline in immune function is no longer the focal point for AIDS therapy, but has evolved to an emphasis on maximizing the potential for immune regeneration. Improved control of HIV replication has diminished, albeit unevenly, the frequency of AIDS-related malignancies and has altered the focus of hematologic and oncologic interventions in HIV disease. Now, with adoptive cellular therapies and the genetic engineering of cells in the clinical arena, the potential for cellular therapeutics in enhancing immune restoration is being tested. These approaches are based on better understanding of the immunobiology of HIV and its impact on hematopoietic tissues.

Expression of isoforms from cloned skeletal and cardiac actin genes.

Some of the molecular events in human muscle parallel in many respects the data already presented regarding the expression or co-expression of certain isoforms of contractile protein genes. Contractile protein isoforms can be studied in a model system based on the genetic engineering of cells in which we introduce contractile protein genes into foreign environments and, once they are expressed, ask what they do to the phenotype of the cell. One can also use a muscle cDNA library to study muscle specific genes and their human chromosomal linkages.KeywordsSatellite CellHuman Skeletal MuscleHuman MyoblastCardiac ActinSarcomeric ActinThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.