Transfection of Babesia duncani: A Genetic Toolbox of this Pathogen to Advance Babesia Biology.

Human babesiosis is a tick-borne disease caused by Babesia pathogens. The disease, which presents with malaria-like symptoms, can be life-threatening, especially in individuals with weakened immune systems and the elderly. The worldwide prevalence of human babesiosis has been gradually rising, prompting alarm among public health experts. In other pathogens, genetic techniques have proven to be valuable tools for conducting functional studies to understand the importance of specific genes in development and pathogenesis as well as to validate novel cellular targets for drug discovery. Genetic manipulation methods have been established for several non-human Babesia and Theileria species and, more recently, have begun to be developed for human Babesia parasites. We have previously reported the development of a method for genetic manipulation of the human pathogen Babesia duncani. This method is based on positive selection using the hDHFR gene as a selectable marker, whose expression is regulated by the ef-1aB promoter, along with homology regions that facilitate integration into the gene of interest through homologous recombination. Herein, we provide a detailed description of the steps needed to implement this strategy in B. duncani to study gene function. It is anticipated that the implementation of this method will significantly improve our understanding of babesiosis and facilitate the development of novel and more effective therapeutic strategies for the treatment of human babesiosis. Key features This protocol provides an effective means of transfection of B. duncani, enabling genetic manipulation and editing to gain further insights into its biology and pathogenesis. The protocol outlined here for the electroporation of B. duncani represents an advancement over previous methods used for B. bovis [1]. Improvements include higher volume of culture used during the electroporation step and an enhancement in the number of electroporation pulses. These modifications likely enhance the efficiency of gene editing in B. duncani, allowing for quicker and more effective selection of transgenic parasites.

Optimized Workflow from Gene to Product.

This chapter outlines the workflow using the ExpiSf™ Expression System designed for high-density infection of suspension ExpiSf9™ cells. The system utilizes a chemically defined, serum-free, protein-free, and animal origin free medium, making it suitable for recombinant protein expression experiments. The ExpiSf™ chemically defined medium allows efficient transfection and baculovirus production directly within the same culture medium. The ExpiSf™ Expression System Starter Kit provides all necessary components, including cells, culture medium, and reagents needed to infect one (1) liter of cell culture. The system's versatility and animal origin free nature make it a valuable tool for various protein expression studies and biotechnological applications.

Construction of Recombinant Baculoviruses.

The success of using the insect cell-baculovirus expression technology (BEST) relies on the efficient construction of recombinant baculovirus with genetic stability and high productivity, ideally within a short time period. Generation of recombinant baculoviruses requires the transfection of insect cells, harvesting of recombinant baculovirus pools, isolation of plaques, and the expansion of baculovirus stocks for their use for recombinant protein production. Moreover, many options exist for selecting the genetic elements to be present in the recombinant baculovirus. This chapter describes the most commonly used homologous recombination systems for the production of recombinant baculoviruses, as well as strategies to maximize generation efficiency and recombinant protein or baculovirus production. The key steps for generating baculovirus stocks and troubleshooting strategies are described.

RNAi-Mediated Silencing in the Insect Cell-Baculovirus Expression System.

RNA interference (RNAi) serves as an indispensable tool for gene function studies and has been substantiated through extensive research for its practical applications in the baculovirus expression vector system (BEVS). This chapter expands the RNAi toolkit in insect cell culture by including small interfering RNA (siRNA) in the protocol, in addition to the conventional use of double-stranded RNA (dsRNA). This chapter also brings attention to key design and reporting considerations, based on Minimum Information About an RNAi Experiment (MIARE) guidelines. Recommendations regarding online tools for dsRNA and siRNA design are provided, along with guidance on choosing suitable methods for measuring silencing outcomes.

Nonviral Platform for Expression of Recombinant Protein in Insect Cells.

Nonviral transfection has been used to express various recombinant proteins, therapeutics, and virus-like particles (VLP) in mammalian and insect cells. Virus-free methods for protein expression require fewer steps for obtaining protein expression by eliminating virus amplification and measuring the infectivity of the virus. The nonviral method uses a nonlytic plasmid to transfect the gene of interest into the insect cells instead of using baculovirus, a lytic system. In this chapter, we describe one of the transfection methods, which uses polyethyleneimine (PEI) as a DNA delivery material into the insect cells to express the recombinant protein in both adherent and suspension cells.

Optimized CRISPR-based knockout in BeWo cells.

CRISPR genome editing is a widely used tool to perturb genes of interest within cells and tissues and can be used as a research tool to study the connection between genotypes and cellular phenotypes. Highly efficient genome editing is limited in certain cell types due to low transfection efficiency or single-cell survivability. This is true for BeWo cells, an in vitro model of placental syncytiotrophoblast cell-cell fusion and hormone secretion. Here we describe an optimized and easy-to-use protocol for knockout in BeWo cells using CRISPR Cas9 ribonucleoprotein (RNP) complexes delivered via electroporation. Further, we describe parameters for successful guide RNA design and how to assess genetic knockouts in BeWo cells so that users can apply this technique to their own genes of interest. We provide a positive control for inducing highly efficient knockout of the cell-cell fusion protein Syncytin-2 (ERVFRD-1) and assessing editing efficiency at this locus. We anticipate that efficient RNP-mediated genetic knockouts in BeWo cells will facilitate the study of new genes involved in cell-cell fusion and hormone secretion in this important cellular model system. Furthermore, this strategy of optimized nucleofection and RNP delivery may be of use in other difficult-to-edit trophoblast cells or could be applied to efficiently deliver transgenes to BeWo cells.

Non-industrial production of therapeutic lentiviral vectors: How to provide vectors to academic CAR-T.

Bringing effective cancer therapy in the form of chimeric antigen receptor technology to untapped markets faces numerous challenges, including a global shortage of therapeutic lentiviral or retroviral vectors on which all current clinical therapies using genetically modified T cells are based. Production of these lentiviral vectors in academic settings in principle opens the way to local production of therapeutic cells, which is the only economically viable approach to make this therapy available to patients in developing countries. The conditions for obtaining and concentrating lentiviral vectors have been optimized and described. The calcium phosphate precipitation method was found to be suitable for transfecting high cell-density cultures, a prerequisite for high titers. We describe protocols for gradually increasing production from 6-well plates to P100 plates, T-175 flasks, and 5-layer stacks while maintaining high titers, >108 transducing units. Concentration experiments using ultracentrifugation revealed the advantage of lower centrifugation speeds compared to competing protocols. The resulting batches of lentiviral vectors had a titer of 1010 infectious particles and were used to transduce primary human T lymphocytes generating chimeric antigen receptor T cells, the quality of which was checked and found potential applicability for treatment.

Degradation of specific glycosaminoglycans improves transfection efficiency and vector production in transient lentiviral vector manufacturing processes.

Both cell surface and soluble extracellular glycosaminoglycans have been shown to interfere with the exogenous nucleic acid delivery efficiency of non-viral gene delivery, including lipoplex and polyplex-mediated transfection. Most gene therapy viral vectors used commercially and in clinical trials are currently manufactured using transient transfection-based bioprocesses. The growing demand for viral vector products, coupled with a global shortage in production capability, requires improved transfection technologies and processes to maximise process efficiency and productivity. Soluble extracellular glycosaminoglycans were found to accumulate in the conditioned cell culture medium of suspension adapted HEK293T cell cultures, compromising transfection performance and lentiviral vector production. The enzymatic degradation of specific, chondroitin sulphate-based, glycosaminoglycans with chondroitinase ABC was found to significantly enhance transfection performance. Additionally, we report significant improvements in functional lentiviral vector titre when cultivating cells at higher cell densities than those utilised in a control lentiviral vector bioprocess; an improvement that was further enhanced when cultures were supplemented with chondroitinase ABC prior to transfection. A 71.2% increase in functional lentiviral vector titre was calculated when doubling the cell density prior to transfection compared to the existing process and treatment of the high-density cell cultures with 0.1 U/mL chondroitinase ABC resulted in a further 18.6% increase in titre, presenting a method that can effectively enhance transfection performance.

Construction of GSH-responsive polyethyleneimine-based delivery vector for effective gene transfection.

The rise of gene therapy has solved many diseases that cannot be effectively treated by conventional methods. Gene vectors is very important to protect and deliver the therapeutic genes to the target site. Polyethyleneimine (PEI) modified with mannitol could enhance the gene transfection efficiency reported by our group previously. In order to further control and improve the effective gene release to action site, disulfide bonds were introduced into mannitol-modified PEI to construct new non-viral gene vectors PeiSM. The degrees of mannitol linking with disulfide bonds were screened. Among them, moderate mannitol-modified polyethyleneimine with disulfide bonds (M-PeiSM) showed the best transfection efficiency, and significantly enhanced long-term systemic transgene expression for 72 hours in vivo even at a single dose administration, and could promote caveolae-mediated uptake through up-regulating the phosphorylation of caveolin-1 and increase the loaded gene release from the nanocomplexes in high GSH intracellular environment. This functionalized gene delivery system can be used as an potential and safe non-viral nanovector for further gene therapy.

Interaction proteomics analysis to provide insight into TFAMoplex-mediated transfection.

In an earlier investigation, our group introduced the TFAMoplex, a transfection agent based on the mitochondrial transcription factor A (TFAM) protein, which complexes DNA into nanoparticles. The original TFAMoplex further contained a bacterial phospholipase to achieve endosomal escape, and the vaccinia-related kinase 1 (VRK1), which significantly boosted the transfection efficiency of the system by an unknown mechanism. This study aims at replacing VRK1 within the TFAMoplex with dynein light chain proteins, specifically RP3, to directly tether the complexes to the dynein motor complex for enhanced cytosolic transport. To confirm the interaction between the dynein complex and the resulting fusion protein, we examined the binding kinetics of TFAM-RP3 to the dynein intermediate chains 1 and 2. Furthermore, we established a proteomics-based assay to compare cytosolic protein interactions of different TFAMoplex variants, including the RP3-modified version and the original VRK1-containing system. In the group of the VRK1-containing TFAMoplex, significant shifts of protein interactors were observed, especially for nucleolar proteins. Leveraging this knowledge, we incorporated one of these nuclear proteins, leucine-rich repeat-containing protein 59 (LRRC59), into the TFAMoplex, resulting in a significant improvement of transfection properties compared to the RP3-modified system and comparable levels versus the original, VRK1-containing version. This study not only advances our comprehension of the TFAMoplex system but also offers broader insights into the potential of protein engineering for designing effective gene delivery systems.

Unraveling mRNA delivery bottlenecks of ineffective delivery vectors by co-transfection with effective carriers.

The messenger RNA (mRNA) SARS-CoV-2 vaccines have demonstrated the therapeutic potential of this novel drug modality. Protein expression is the consequence of a multistep delivery process that relies on proper packaging into nanoparticle carriers to protect the mRNA against degradation enabling effective cellular uptake and endosomal release and liberating the mRNA in the cytosol. Bottlenecks along this route remain challenging to pinpoint. Although methods to assess endosomal escape of carriers have been developed, versatile strategies to identify bottlenecks along the delivery trajectory are missing. Here, it is shown that co-incubating an inefficient nanoparticle formulation with an efficient one solves this problem. Cells were co-incubated with mRNA nanoparticles formed with either the efficient cell-penetrating peptide (CPP) PepFect14 or the inefficient CPP nona-arginine (R9). Co-transfection enhanced cellular uptake and endosomal escape of R9-formulated mRNA, resulting in protein expression, demonstrating that both vectors enter cells along the same route. In addition, cells were transfected with a galectin-9-mCherry fusion protein to detect endosomal rupture. Remarkably, despite endosomal release, mRNA remained confined to punctate structures, identifying mRNA liberation as a further bottleneck. In summary, co-transfection offers a rapid means to identify bottlenecks in cytosolic mRNA delivery, supporting the rational design and optimization of intracellular mRNA delivery systems.

Realizing time-staggered expression of nucleic acid-encoded proteins by co-delivery of messenger RNA and plasmid DNA on a single nanocarrier.

Co-delivery of different protein-encoding polynucleotide species with varying expression kinetics of their therapeutic product will become a prominent requirement in the realm of combined nucleic acid(NA)-based therapies in the upcoming years. The current study explores the capacity for time-staggered expression of encoded proteins by simultaneous delivery of plasmid DNA (pDNA) in the core and mRNA on the shell of the same nanocarrier. The core is based on a Gelatin Type A-pDNA coacervate, thermally stabilized to form an irreversible nanogel stable enough for the deposition of cationic coats namely, protamine sulfate or LNP-related lipid mixtures. Only the protamine-coated nanocarriers remained colloidally stable following mRNA loading and could successfully co-transfect murine dendritic cell line DC2.4 with fluorescent reporter mRNA(mCherry) and pDNA (pAmCyan1). Further investigation of the protamine-coated nanosystem only, the transfection efficiency (percentage of transfected cells) and level of protein expression (mean fluorescence intensity, MFI) of mRNA and pDNA, simultaneously delivered by the same nanocarrier, were compared and kinetically assessed over 48 h in DC2.4 using flow cytometry. The onset of transfection for both nucleotides was initially delayed, with levels < 5% at 6 h. Thereafter, mRNA transfection reached 90% after 24 h and continued to slightly increase until 48 h. In contrast, pDNA transfection was clearly slower, reaching approximately 40% after 24 h, but continuing to increase to reach 94% at 48 h. The time course of protein expression (represented by MFI) for both NAs essentially followed that of transfection. Model-independent as well as model-dependent kinetic parameters applied to the data further confirmed such time-staggered expression of the two NA's where mRNA's rate of transfection and protein expression initially exceeded those of pDNA in the first 24 h of the experiment whereas the opposite was true during the second 24 h of the experiment where pDNA displayed the higher response rates. We expect that innovative nanocarriers capable of time-staggered co-delivery of different nucleotides could open new perspectives for multi-dosing, pulsatile or sustained expression of nucleic acid-based therapeutics in protein replacement, vaccination, and CRISPR-mediated gene editing scenarios.

Plasmid Delivery and Single-Cell Plasmid Expression Analysis for CRISPR/dCas9-Based Epigenetic Editing.

To fully exploit the potentials of reprogramming the epigenome through CRISPR/dCas9 systems for epigenetic editing, there is a growing need for improved transfection methods. With the utilization of constructs often with large sizes and the wide array of cell types used to read out the effect of epigenetic editing in different biological applications, it is evident that ongoing optimalization of transfection protocols tailored to each specific experimental setup is essential. Whether the goal is the production of viral particles using human embryonic kidney (HEK) cells or the direct examination of epigenomic modifications in the target cell type, continuous refinement of transfection methods is crucial. In the hereafter outlined protocol, we focus on optimization of transfection protocols by comparing different reagents and methods, creating a streamlined setup for transfection efficiency optimization in cultured mammalian cells. Our protocol provides a comprehensive overview of flow cytometry analysis following transfection not just to improve transfection efficiency but also to assess the expression level of the utilized construct. We showcase our transfection protocol optimization using HEK293T Lenti-X™ and breast cancer MCF-7 cell lines, using a single-guide RNA-containing plasmid. Specifically, we incorporate heat shock treatment for increased transfection efficiency of the MCF-7 cell line. Our detailed optimization protocol for efficient plasmid delivery and measurement of single-cell plasmid expression provides a comprehensive instruction for assessing both transient and sustained effects of epigenetic reprogramming.

Gene silencing in the aedine cell lines C6/36 and U4.4 using long double-stranded RNA.

BACKGROUND: RNA interference (RNAi) is a target-specific gene silencing method that can be used to determine gene functions and investigate host-pathogen interactions, as well as facilitating the development of ecofriendly pesticides. Commercially available transfection reagents (TRs) can improve the efficacy of RNAi. However, we currently lack a product and protocol for the transfection of insect cell lines with long double-stranded RNA (dsRNA). METHODS: We used agarose gel electrophoresis to determine the capacity of eight TRs to form complexes with long dsRNA. A CellTiter-Glo assay was then used to assess the cytotoxicity of the resulting lipoplexes. We also measured the cellular uptake of dsRNA by fluorescence microscopy using the fluorophore Cy3 as a label. Finally, we analyzed the TRs based on their transfection efficacy and compared the RNAi responses of Aedes albopictus C6/36 and U4.4 cells by knocking down an mCherry reporter Semliki Forest virus in both cell lines. RESULTS: The TRs from Biontex (K4, Metafectene Pro, and Metafectene SI+) showed the best complexing capacity and the lowest dsRNA:TR ratio needed for complete complex formation. Only HiPerFect was unable to complex the dsRNA completely, even at a ratio of 1:9. Most of the complexes containing mCherry-dsRNA were nontoxic at 2 ng/µL, but Lipofectamine 2000 was toxic at 1 ng/µL in U4.4 cells and at 2 ng/µL in C6/36 cells. The transfection of U4.4 cells with mCherry-dsRNA/TR complexes achieved significant knockdown of the virus reporter. Comparison of the RNAi response in C6/36 and U4.4 cells suggested that C6/36 cells lack the antiviral RNAi response because there was no significant knockdown of the virus reporter in any of the treatments. CONCLUSIONS: C6/36 cells have an impaired RNAi response as previously reported. This investigation provides valuable information for future RNAi experiments by showing how to mitigate the adverse effects attributed to TRs. This will facilitate the judicious selection of TRs and transfection conditions conducive to RNAi research in mosquitoes.

Intercellular transfer of plasmid DNA between in vitro cultured HEK293 cells following transient transfection.

Gene overexpression by transient transfection of in vitro cultured model cell lines with plasmid DNA is a commonly used method for studying molecular aspects of human biology and pathobiology. However, there is accumulating evidence suggesting that human cells may actively secrete fragments of DNA and the implications of this phenomenon for in vitro cultured cells transiently transfected with foreign nucleic acids has been overlooked. Therefore, in the current study we investigated whether a cell-to-cell transmission of acquired plasmid DNA takes place in a commonly used human cell line model. We transiently transfected HEK293 cells with EGFP encoding plasmids to serve as donor cells and either co-cultured these with stably mCherry expressing recipient cells in different set-ups or transferred their culture medium to the recipient cells. We found that recipient cells produced EGFP after being co-cultured with donor cells but not when they were exposed to their culture medium. The employment of different co-culture set-ups excluded that the observed effect stemmed from technical artefacts and provided evidence that an intercellular plasmid transfer takes place requiring physical proximity between living cells. This phenomenon could represent a significant biological artefact for certain studies such as those addressing protein transmissions in prion diseases.

Development and validation of novel keloid-derived immortalized fibroblast cell lines.

Keloids are a common connective tissue disorder with an ill-understood etiopathogenesis and no effective treatment. This is exacerbated because of the absence of an animal model. Patient-derived primary keloid cells are insufficient as they age through passaging and have a limited supply. Therefore, there is an unmet need for development of a cellular model that can consistently and faithfully represent keloid's pathognomic features. In view of this, we developed keloid-derived immortalized fibroblast (KDIF) cell lines from primary keloid fibroblasts (PKF) by transfecting the human telomerase reverse transcriptase (hTERT) gene. The TERT gene encodes the catalytic subunit of the telomerase enzyme, which is responsible for maintaining the cellular replicative potential (cellular immortalization). Primary fibroblasts from keloid-specific lesional (peripheral, middle, and top) as well as extralesional sites were isolated and evaluated for cell line development and comparative cellular characteristics by employing qRT-PCR and immunofluorescence staining. Moreover, the immortalized behavior of KDIF cell lines was evaluated by comparing with cutaneous fibrosarcoma and dermatofibrosarcoma protuberans cell lines. Stable KDIF cell lines with elevated expression of hTERT exhibited the cellular characteristics of site-specific keloid fibroblasts. Histochemical staining for ß-galactosidase revealed a significantly lower number of ß-gal-positive cells in all three KDIF cell lines compared with that in PKFs. The cell growth curve pattern was studied over 10 passages for all three KDIF cell lines and was compared with the control groups. The results showed that all three KDIF cell lines grew significantly faster and obtained a fast growing characteristic as compared to primary keloid and normal fibroblasts. Phenotypic behavior in growth potential is an indication of hTERT-mediated immortalized transformation. Cell migration analysis revealed that the top and middle KDIF cell lines exhibited similar migration trend as site-specific PKFs. Notably, peripheral KDIF cell line showed significantly enhanced cell migration in comparison to the primary peripheral fibroblasts. All KDIF cell lines expressed Collagen I protein as a keloid-associated fibrotic marker. Functional testing with triamcinolone inhibited cell migration in KDIF. ATCC short tandem repeat profiling validated the KDIF as keloid representative cell line. In summary, we provide the first novel KDIF cell lines. These cell lines overcome the limitations related to primary cell passaging and tissue supply due to immortalized features and present an accessible and consistent experimental model for keloid research.

Plasmodium falciparum CLAG Paralogs All Traffic to the Host Membrane but Knockouts Have Distinct Phenotypes.

Malaria parasites increase their host erythrocyte's permeability to obtain essential nutrients from plasma and facilitate intracellular growth. In the human Plasmodium falciparum pathogen, this increase is mediated by the plasmodial surface anion channel (PSAC) and has been linked to CLAG3, a protein integral to the host erythrocyte membrane and encoded by a member of the conserved clag multigene family. Whether paralogs encoded by other clag genes also insert at the host membrane is unknown; their contributions to PSAC formation and other roles served are also unexplored. Here, we generated transfectant lines carrying epitope-tagged versions of each CLAG. Each paralog is colocalized with CLAG3, with concordant trafficking via merozoite rhoptries to the host erythrocyte membrane of newly invaded erythrocytes. Each also exists within infected cells in at least two forms: an alkaline-extractable soluble form and a form integral to the host membrane. Like CLAG3, CLAG2 has a variant region cleaved by extracellular proteases, but CLAG8 and CLAG9 are protease resistant. Paralog knockout lines, generated through CRISPR/Cas9 transfection, exhibited uncompromised growth in PGIM, a modified medium with higher physiological nutrient levels; this finding is in marked contrast to a recently reported CLAG3 knockout parasite. CLAG2 and CLAG8 knockout lines exhibited compensatory increases in the transcription of the remaining clags and associated rhoph genes, yielding increased PSAC-mediated uptake for specific solutes. We also report on the distinct transport properties of these knockout lines. Similar membrane topologies at the host membrane are consistent with each CLAG paralog contributing to PSAC, but other roles require further examination.

Cost-Efficient Expression of Human Cardiac Myosin Heavy Chain in C2C12 Cells with a Non-Viral Transfection Reagent.

Production of functional myosin heavy chain (MHC) of striated muscle myosin II for studies of isolated proteins requires mature muscle (e.g., C2C12) cells for expression. This is important both for fundamental studies of molecular mechanisms and for investigations of deleterious diseases like cardiomyopathies due to mutations in the MHC gene (MYH7). Generally, an adenovirus vector is used for transfection, but recently we demonstrated transfection by a non-viral polymer reagent, JetPrime. Due to the rather high costs of JetPrime and for the sustainability of the virus-free expression method, access to more than one transfection reagent is important. Here, we therefore evaluate such a candidate substance, GenJet. Using the human cardiac ß-myosin heavy chain (ß-MHC) as a model system, we found effective transfection of C2C12 cells showing a transfection efficiency nearly as good as with the JetPrime reagent. This was achieved following a protocol developed for JetPrime because a manufacturer-recommended application protocol for GenJet to transfect cells in suspension did not perform well. We demonstrate, using in vitro motility assays and single-molecule ATP turnover assays, that the protein expressed and purified from cells transfected with the GenJet reagent is functional. The purification yields reached were slightly lower than in JetPrime-based purifications, but they were achieved at a significantly lower cost. Our results demonstrate the sustainability of the virus-free method by showing that more than one polymer-based transfection reagent can generate useful amounts of active MHC. Particularly, we suggest that GenJet, due to its current ~4-fold lower cost, is useful for applications requiring larger amounts of a given MHC variant.

Use of Transient Transfection for cGMP Manufacturing of eOD-GT8 60mer, a Self-Assembling Nanoparticle Germline-Targeting HIV-1 Vaccine Candidate.

We describe the current Good Manufacturing Practice (cGMP) production and subsequent characterization of eOD-GT8 60mer, a glycosylated self-assembling nanoparticle HIV-1 vaccine candidate and germline targeting priming immunogen. Production was carried out via transient expression in the human embryonic kidney 293 (HEK293) cell line followed by a combination of purification techniques. A large-scale cGMP (200 L) production run yielded 354 mg of the purified eOD-GT8 60mer drug product material, which was formulated at 1 mg/mL in 10% sucrose in phosphate-buffered saline (PBS) at pH 7.2. The clinical trial material was comprehensively characterized for purity, antigenicity, glycan composition, amino acid sequence, and aggregation and by several safety-related tests during cGMP lot release. A comparison of the purified products produced at the 1 L scale and 200 L cGMP scale demonstrated the consistency and robustness of the transient transfection upstream process and the downstream purification strategies. The cGMP clinical trial material was tested in a Phase 1 clinical trial (NCT03547245), is currently being stored at -80 °C, and is on a stability testing program as per regulatory guidelines. The methods described here illustrate the utility of transient transfection for cGMP production of complex products such as glycosylated self-assembling nanoparticles.

Fundamentals and recent advances in the evaluation and management of medullary thyroid carcinoma.

Medullary thyroid carcinoma (MTC) is a rare primary neuroendocrine thyroid carcinoma that is distinct from other thyroid or neuroendocrine cancers. Most cases of MTC are sporadic, although MTC exhibits a high degree of heritability as part of the multiple endocrine neoplasia syndromes. REarranged during Transfection (RET) mutations are the primary oncogenic drivers and advances in molecular profiling have revealed that MTC is enriched in druggable alterations. Surgery at an early stage is the only chance for cure, but many patients present with or develop metastases. C-cell-specific calcitonin trajectory and structural doubling times are critical biomarkers to inform prognosis, extent of surgery, likelihood of residual disease, and need for additional therapy. Recent advances in the role of active surveillance, regionally directed therapies for localized disease, and systemic therapy with multi-kinase and RET-specific inhibitors for progressive/metastatic disease have significantly improved outcomes for patients with MTC.