Supramolecular assembly of polycation/mRNA nanoparticles and in vivo monocyte programming.

Size-dependent phagocytosis is a well-characterized phenomenon in monocytes and macrophages. However, this size effect for preferential gene delivery to these important cell targets has not been fully exploited because commonly adopted stabilization methods for electrostatically complexed nucleic acid nanoparticles, such as PEGylation and charge repulsion, typically arrest the vehicle size below 200 nm. Here, we bridge the technical gap in scalable synthesis of larger submicron gene delivery vehicles by electrostatic self-assembly of charged nanoparticles, facilitated by a polymer structurally designed to modulate internanoparticle Coulombic and van der Waals forces. Specifically, our strategy permits controlled assembly of small poly(ß-amino ester)/messenger ribonucleic acid (mRNA) nanoparticles into particles with a size that is kinetically tunable between 200 and 1,000 nm with high colloidal stability in physiological media. We found that assembled particles with an average size of 400 nm safely and most efficiently transfect monocytes following intravenous administration and mediate their differentiation into macrophages in the periphery. When a CpG adjuvant is co-loaded into the particles with an antigen mRNA, the monocytes differentiate into inflammatory dendritic cells and prime adaptive anticancer immunity in the tumor-draining lymph node. This platform technology offers a unique ligand-independent, particle-size-mediated strategy for preferential mRNA delivery and enables therapeutic paradigms via monocyte programming.

Data-balanced transformer for accelerated ionizable lipid nanoparticles screening in mRNA delivery.

Despite the widespread use of ionizable lipid nanoparticles (LNPs) in clinical applications for messenger RNA (mRNA) delivery, the mRNA drug delivery system faces an efficient challenge in the screening of LNPs. Traditional screening methods often require a substantial amount of experimental time and incur high research and development costs. To accelerate the early development stage of LNPs, we propose TransLNP, a transformer-based transfection prediction model designed to aid in the selection of LNPs for mRNA drug delivery systems. TransLNP uses two types of molecular information to perceive the relationship between structure and transfection efficiency: coarse-grained atomic sequence information and fine-grained atomic spatial relationship information. Due to the scarcity of existing LNPs experimental data, we find that pretraining the molecular model is crucial for better understanding the task of predicting LNPs properties, which is achieved through reconstructing atomic 3D coordinates and masking atom predictions. In addition, the issue of data imbalance is particularly prominent in the real-world exploration of LNPs. We introduce the BalMol block to solve this problem by smoothing the distribution of labels and molecular features. Our approach outperforms state-of-the-art works in transfection property prediction under both random and scaffold data splitting. Additionally, we establish a relationship between molecular structural similarity and transfection differences, selecting 4267 pairs of molecular transfection cliffs, which are pairs of molecules that exhibit high structural similarity but significant differences in transfection efficiency, thereby revealing the primary source of prediction errors. The code, model and data are made publicly available at https://github.com/wklix/TransLNP.

Inhaled mRNA nanoparticles dual-targeting cancer cells and macrophages in the lung for effective transfection.

Messenger RNA (mRNA)-based therapeutics are transforming the landscapes of medicine, yet targeted delivery of mRNA to specific cell types while minimizing off-target accumulation remains challenging for mRNA-mediated therapy. In this study, we report an innovative design of a cationic lipid- and hyaluronic acid-based, dual-targeted mRNA nanoformulation that can display the desirable stability and efficiently transfect the targeted proteins into lung tissues. More importantly, the optimized dual-targeted mRNA nanoparticles (NPs) can not only accumulate primarily in lung tumor cells and inflammatory macrophages after inhalation delivery but also efficiently express any desirable proteins (e.g., p53 tumor suppressor for therapy, as well as luciferase and green fluorescence protein for imaging as examples in this study) and achieve efficacious lung tissue transfection in vivo. Overall, our findings provide proof-of-principle evidence for the design and use of dual-targeted mRNA NPs in homing to specific cell types to up-regulate target proteins in lung tissues, which may hold great potential for the future development of mRNA-based inhaled medicines or vaccines in treating various lung-related diseases.

Cargo-selective and adaptive delivery of nucleic acid therapeutics by bola-amphiphilic dendrimers.

Nucleic acid therapeutics are becoming an important drug modality, offering the unique opportunity to address "undruggable" targets, respond rapidly to evolving pathogens, and treat diseases at the gene level for precision medicine. However, nucleic acid therapeutics have poor bioavailability and are chemolabile and enzymolabile, imposing the need for delivery vectors. Dendrimers, by virtue of their well-defined structure and cooperative multivalence, represent precision delivery systems. We synthesized and studied bola-amphiphilic dendrimers for cargo-selective and on-demand delivery of DNA and small interfering RNA (siRNA), both important nucleic acid therapeutics. Remarkably, superior performances were achieved for siRNA delivery with the second-generation dendrimer, yet for DNA delivery with the third generation. We systematically studied these dendrimers with regard to cargo binding, cellular uptake, endosomal release, and in vivo delivery. Differences in size both of the dendrimers and their nucleic acid cargos impacted the cooperative multivalent interactions for cargo binding and release, leading to cargo-adaptive and selective delivery. Moreover, both dendrimers harnessed the advantages of lipid and polymer vectors, while offering nanotechnology-based tumor targeting and redox-responsive cargo release. Notably, they allowed tumor- and cancer cell-specific delivery of siRNA and DNA therapeutics for effective treatment in different cancer models, including aggressive and metastatic malignancies, outperforming the currently available vectors. This study provides avenues to engineer tailor-made vectors for nucleic acid delivery and precision medicine.

Streptococcus pyogenes Cas9 ribonucleoprotein delivery for efficient, rapid and marker-free gene editing in Trypanosoma and Leishmania.

Kinetoplastids are unicellular eukaryotic flagellated parasites found in a wide range of hosts within the animal and plant kingdoms. They are known to be responsible in humans for African sleeping sickness (Trypanosoma brucei), Chagas disease (Trypanosoma cruzi), and various forms of leishmaniasis (Leishmania spp.), as well as several animal diseases with important economic impact (African trypanosomes, including Trypanosoma congolense). Understanding the biology of these parasites necessarily implies the ability to manipulate their genomes. In this study, we demonstrate that transfection of a ribonucleoprotein complex, composed of recombinant Streptococcus pyogenes Cas9 (SpCas9) and an in vitro-synthesized guide RNA, results in rapid and efficient genetic modifications of trypanosomatids, in marker-free conditions. This approach was successfully developed to inactivate, delete, and mutate candidate genes in various stages of the life cycle of T. brucei and T. congolense, and Leishmania promastigotes. The functionality of SpCas9 in these parasites now provides, to the research community working on these parasites, a rapid and efficient method of genome editing, without requiring plasmid construction and selection by antibiotics but requires only cloning and PCR screening of the clones. Importantly, this approach is adaptable to any wild-type parasite.

Optimizing miRNA transfection for screening in precision cut lung slices.

Precision cut lung slices(PCLS) are complex 3D lung tissue models, which preserve the native microenvironment, including cell diversity and cell-matrix interactions. They are an innovative ex vivo platform that allows studying disease as well as the effects of therapeutic agents or regulatory molecules(e.g. miRNA). The aim of our study was to develop a protocol to transfect PCLS with miRNA using lipid nanoparticles (LNPs) to enable higher throughput screening of miRNA, obviating the need for custom stabilization and internalization approaches. 4mm diameter PCLS were generated using agarose-filled rodent lungs and a vibratome. TYE665 labelled scrambled miRNA was used to evaluate transfection efficacy of six different commercially available LNPs. Transfection efficacy was visualised using live high content fluorescence microscopy, followed by higher resolution confocal fluorescence microscopy in fixed PCLS. Metabolic activity and cellular damage were assessed using WST-1 and lactate dehydrogenase(LDH) release. Using a live staining kit containing a cell membrane impermeant nuclear dye, RedDot2TM, we established that cellular membranes in PCLS are permeable in the initial 24 hours of slicing but diminished thereafter. Therefore, all transfection experiments occurred at least 24 hours after slicing. All six commercially available LNPs enabled transfection without inducing significant cytotoxicity or impaired metabolic function. However, RNAiMAX and INTERFERin led to increases in transfection efficacy as compared to other LNPs, with detection possible as low as 25nM. Therefore, LNP-based transfection of miRNA is possible and can be visualized in live or fixed PCLS, enabling future higher throughput studies using diverse miRNAs.

Establishment of a novel minigenome system for the identification of drugs targeting Nipah virus replication.

Nipah virus (NiV) is a deadly zoonotic pathogen with high potential to cause another pandemic. Owing to biosafety concerns, studies on living NiV must be performed in biosafety level 4 (BSL-4) laboratories, which greatly hinders the development of anti-NiV drugs. To overcome this issue, minigenome systems have been developed to study viral replication and screen for antiviral drugs. This study aimed to develop two minigenome systems (transient and stable expression) based on a helper cell line expressing the NiV P, N and L proteins required to initiate NiV RNA replication. Stable minigenome cells were resistant to ribavirin, remdesivir and favipiravir but sensitive to interferons. Cells of the transient replication system were sensitive to ribavirin and favipiravir and suitable for drug screening. Our study demonstrates a feasible and effective platform for studying NiV replication and shows great potential for high-throughput drug screening in a BSL-2 laboratory environment.

Tailoring transfection for cardiomyocyte cell lines: balancing efficiency and toxicity in lipid versus polymer-based transfection methods in H9c2 and HL-1 cells.

Cardiovascular research relies heavily on the veracity of in vitro cardiomyocyte models, with H9c2 and HL-1 cell lines at the forefront due to their cardiomyocyte-like properties. However, the variability stemming from nonstandardized culturing and transfection methods poses a significant challenge to data uniformity and reliability. In this study, we introduce meticulously crafted protocols to enhance the culture and transfection of H9c2 and HL-1 cells, emphasizing the reduction of cytotoxic effects while improving transfection efficiency. Through the examination of polymer-based and lipid-based transfection methods, we offer a comparative analysis that underscores the heightened efficiency and reduced toxicity of these approaches. Our research provides an extensive array of step-by-step procedures designed to foster robust cell cultures and outlines troubleshooting practices to rectify issues of low transfection rates. We discuss the merits and drawbacks of both transfection techniques, equipping researchers with the knowledge to choose the most fitting method for their experimental goals. By offering a definitive guide to these cell lines' culturing and transfection, our work seeks to set a new standard in procedural consistency, ensuring that the cardiovascular research community can achieve more dependable and reproducible results, thereby pushing the boundaries of current methodologies toward impactful clinical applications.NEW & NOTEWORTHY We have developed standardized protocols that significantly reduce cytotoxicity and enhance transfection efficiency in H9c2 and HL-1 cardiomyocyte cell lines. Our detailed comparative analysis of polymer-based and lipid-based transfection methods has identified optimized approaches with superior performance. Accompanying these protocols are comprehensive troubleshooting strategies to address common issues related to low transfection rates. Implementing these protocols is expected to yield more consistent and reproducible results, driving the field of cardiovascular research toward impactful clinical breakthroughs.

A solution for highly efficient electroporation of primary cytotoxic T lymphocytes.

BACKGROUND: Cytotoxic T lymphocytes (CTLs) are central players in the adaptive immune response. Their functional characterization and clinical research depend on efficient and reliable transfection. Although various methods have been utilized, electroporation remains the preferred technique for transient gene over-expression. However, the efficiency of electroporation is reduced for human and mouse primary CTLs. Lonza offers kits that effectively improve plasmid DNA transfection quality. Unfortunately, the removal of key components of the cell recovery medium considerably reduced the efficiency of their kit for CTLs. Our aim was to develop a new recovery medium to be used with Lonza's Nucleofector system that would significantly enhance transfection rates. RESULTS: We assessed the impact of different media in which the primary CTLs were placed to recover after electroporation on cell survival, transfection rate and their ability to form an immunological synapse and to perform exocytosis. We transfected the cells with pmax-GFP and large constructs encoding for either CD81-super ecliptic pHluorin or granzyme B-pHuji. The comparison of five different media for mouse and two for human CTLs demonstrated that our new recovery medium composed of Opti-MEM-GlutaMAX supplemented with HEPES, DMSO and sodium pyruvate gave the best result in cell survival (> 50%) and transfection rate (> 30 and 20% for mouse and human cells, respectively). More importantly, the functionality of CTLs was at least twice as high as with the original Lonza recovery medium. In addition, our RM significantly improved transfection efficacy of natural killer cells that are notoriously hard to electroporate. CONCLUSION: Our results show that successful transfection depends not only on the electroporation medium and pulse sequence but also on the medium applied for cell recovery. In addition, we have reduced our reliance on proprietary products by designing an effective recovery medium for both mouse and human primary CTLs and other lymphocytes that can be easily implemented by any laboratory. We expect that this recovery medium will have a significant impact on both fundamental and applied research in immunology.

Efficient and scalable gene delivery method with easily generated cationic carbon dots.

Gene delivery is a complex process with several challenges when attempting to incorporate genetic material efficiently and safely into target cells. Some of the key challenges include not only efficient cellular uptake and endosomal escape to ensure that the genetic material can exert its effect but also minimizing the toxicity of the delivery system, which is vital for safe gene delivery. Of importance, if gene delivery systems are intended for biomedical applications or clinical use, they must be scalable and easy and affordable to manufacture to meet the demand. Here, we show an efficient gene delivery method using a combination of carbon dots coated by PEI through electrostatic binding to easily generate cationic carbon dots. We show a biofunctional approach to generate optimal cationic carbon dots (CCDs) that can be scaled up to meet specific transfection demands. CCDs improve cell viability and increase transfection efficiency four times over the standard of PEI polyplexes. Generated CCDs enabled the challenging transfection protocol to produce retroviral vectors via cell cotransfection of three different plasmids into packing cells, showing not only high efficiency but also functionality of the gene delivery, tested as the capacity to produce infective retroviral particles.

Gene Delivery From Granular Scaffolds for Tunable Biologics Manufacturing.

The understanding of the molecular basis for disease has generated a myriad of therapeutic biologics, including therapeutic proteins, antibodies, and viruses. However, the promise that biologics can resolve currently incurable diseases hinges in their manufacturability. These therapeutics require that their genetic material be introduced to mammalian cells such that the cell machinery can manufacture the biological components. These are then purified, validated, and packaged. Most manufacturing uses batch processes that collect the biologic a few days following genetic modification, due to toxicity or difficulty in separating product from cells in a continuous operation, limiting the amount of biologic that can be produced and resulting in yearlong backlogs. Here, a scaffold-based approach for continuous biologic manufacturing is presented, with sustained production of active antibodies and viruses for 30 days. The use of scaffold-based biologic production enabled perfusion-based bioreactors to be used, which can be incorporated into a fully continuous process.

Heterochiral modifications enhance robustness and function of DNA in living human cells.

Oligonucleotide therapeutics are becoming increasingly important as more are approved by the FDA, both for treatment and vaccination. Similarly, dynamic DNA nanotechnology is a promising technique that can be used to sense exogenous input molecules or endogenous biomarkers and integrate the results of multiple sensing reactions in situ via a programmed cascade of reactions. The combination of these two technologies could be highly impactful in biomedicine by enabling smart oligonucleotide therapeutics that can autonomously sense and respond to a disease state. A particular challenge, however, is the limited lifetime of standard nucleic acid components in living cells and organisms due to degradation by endogenous nucleases. In this work, we address this challenge by incorporating mirror-image, ʟ-DNA nucleotides to produce heterochiral "gapmers". We use dynamic DNA nanotechnology to show that these modifications keep the oligonucleotide intact in living human cells for longer than an unmodified strand. To this end, we used a sequential transfection protocol for delivering multiple nucleic acids into living human cells while providing enhanced confidence that subsequent interactions are actually occurring within the cells. Taken together, this work advances the state of the art of ʟ-nucleic acid protection of oligonucleotides and DNA circuitry for applications in vivo.

Recent Advancements in Electroporation Technologies: From Bench to Clinic.

Over the past decade, the increased adoption of electroporation-based technologies has led to an expansion of clinical research initiatives. Electroporation has been utilized in molecular biology for mammalian and bacterial transfection; for food sanitation; and in therapeutic settings to increase drug uptake, for gene therapy, and to eliminate cancerous tissues. We begin this article by discussing the biophysics required for understanding the concepts behind the cell permeation phenomenon that is electroporation. We then review nano- and microscale single-cell electroporation technologies before scaling up to emerging in vivo applications.

CGMP Compliant Microfluidic Transfection of Induced Pluripotent Stem Cells for CRISPR-Mediated Genome Editing.

Inherited retinal degeneration is a term used to describe heritable disorders that result from the death of light sensing photoreceptor cells. Although we and others believe that it will be possible to use gene therapy to halt disease progression early in its course, photoreceptor cell replacement will likely be required for patients who have already lost their sight. While advances in autologous photoreceptor cell manufacturing have been encouraging, development of technologies capable of efficiently delivering genome editing reagents to stem cells using current good manufacturing practices (cGMP) are needed. Gene editing reagents were delivered to induced pluripotent stem cells (iPSCs) using a Zephyr microfluidic transfection platform (CellFE). CRISPR-mediated cutting was quantified using an endonuclease assay. CRISPR correction was confirmed via digital PCR and Sanger sequencing. The resulting corrected cells were also karyotyped and differentiated into retinal organoids. We describe use of a novel microfluidic transfection platform to correct, via CRISPR-mediated homology-dependent repair (HDR), a disease-causing NR2E3 mutation in patient-derived iPSCs using cGMP compatible reagents and approaches. We show that the resulting cell lines have a corrected genotype, exhibit no off-target cutting, retain pluripotency and a normal karyotype and can be differentiated into retinal tissue suitable for transplantation. The ability to codeliver CRISPR/Cas9 and HDR templates to patient-derived iPSCs without using proprietary transfection reagents will streamline manufacturing protocols, increase the safety of resulting cell therapies, and greatly reduce the regulatory burden of clinical trials.

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.

A multiplexed microfluidic continuous-flow electroporation system for efficient cell transfection.

Cellular therapies have the potential to advance treatment for a broad array of diseases but rely on viruses for genetic reprogramming. The time and cost required to produce viruses has created a bottleneck that constricts development of and access to cellular therapies. Electroporation is a non-viral alternative for genetic reprogramming that bypasses these bottlenecks, but current electroporation technology suffers from low throughput, tedious optimization, and difficulty scaling to large-scale cell manufacturing. Here, we present an adaptable microfluidic electroporation platform with the capability for rapid, multiplexed optimization with 96-well plates. Once parameters are optimized using small volumes of cells, transfection can be seamlessly scaled to high-volume cell manufacturing without re-optimization. We demonstrate optimizing transfection of plasmid DNA to Jurkat cells, screening hundreds of different electrical waveforms of varying shapes at a speed of ~3 s per waveform using ~20 µL of cells per waveform. We selected an optimal set of transfection parameters using a low-volume flow cell. These parameters were then used in a separate high-volume flow cell where we obtained similar transfection performance by design. This demonstrates an alternative non-viral and economical transfection method for scaling to the volume required for producing a cell therapy without sacrificing performance. Importantly, this transfection method is disease-agnostic with broad applications beyond cell therapy.

Auto-transduction in lentiviral vector bioprocessing: A quantitative assessment and a novel inhibition strategy.

Lentiviral vectors are highly efficient gene delivery vehicles used extensively in the rapidly growing field of cell and gene therapy. Demand for efficient, large-scale, lentiviral vector bioprocessing is growing as more therapies reach late-stage clinical trials and are commercialized. However, despite substantial progress, several process inefficiencies remain. The unintended auto-transduction of viral vector-producing cells by newly synthesized lentiviral vector particles during manufacturing processes constitutes one such inefficiency which remains largely unaddressed. In this study, we determined that over 60% of functional lentiviral vector particles produced during an upstream production process were lost to auto-transduction, highlighting a major process inefficiency likely widespread within the industry. Auto-transduction of cells by particles pseudotyped with the widely used vesicular stomatitis virus G protein was inhibited via the adoption of a reduced extracellular pH during vector production, impairing the ability of the vector to interact with its target receptor. Employing a posttransfection pH shift to pH 6.7-6.8 resulted in a sevenfold reduction in vector genome integration events, arising from lentiviral vector-mediated transduction, within viral vector-producing cell populations and ultimately resulted in improved lentiviral vector production kinetics. The proposed strategy is scalable and cost-effective, providing an industrially relevant approach to improve lentiviral vector production efficiencies.

Multidose transient transfection of human embryonic kidney 293 cells modulates recombinant adeno-associated virus2/5 Rep protein expression and influences the enrichment fraction of filled capsids.

Recombinant adeno-associated virus (rAAV) is a commonly used in vivo gene therapy vector because of its nonpathogenicity, long-term transgene expression, broad tropism, and ability to transduce both dividing and nondividing cells. However, rAAV vector production via transient transfection of mammalian cells typically yields a low fraction of filled-to-total capsids (~1%-30% of total capsids produced). Analysis of our previously developed mechanistic model for rAAV2/5 production attributed these low fill fractions to a poorly coordinated timeline between capsid synthesis and viral DNA replication and the repression of later phase capsid formation by Rep proteins. Here, we extend the model by quantifying the expression dynamics of total Rep proteins and their influence on the key steps of rAAV2/5 production using a multiple dosing transfection of human embryonic kidney 293 (HEK293) cells. We report that the availability of preformed empty capsids and viral DNA copies per cell are not limiting to the capsid-filling reaction. However, optimal expression of Rep proteins (<240 ± 13 ag per cell) enables enrichment of the filled capsid population (>12% of total capsids/cell) upstream. Our analysis suggests increased enrichment of filled capsids via regulating the expression of Rep proteins is possible but at the expense of per cell capsid titer in a triple plasmid transfection. Our study reveals an intrinsic limitation of scaling rAAV2/5 vector genome (vg) production and underscores the need for approaches that allow for regulating the expression of Rep proteins to maximize vg titer per cell upstream.

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.

Molecular genetics, therapeutics and RET inhibitor resistance for medullary thyroid carcinoma and future perspectives.

Medullary thyroid carcinoma (MTC) is a rare type of thyroid malignancy that accounts for approximately 1-2% of all thyroid cancers (TCs). MTC include hereditary and sporadic cases, the former derived from a germline mutation of rearrangement during transfection (RET) proto-oncogene, whereas somatic RET mutations are frequently present in the latter. Surgery is the standard treatment for early stage MTC, and the 10-year survival rate of early MTC is over 80%. While for metastatic MTC, chemotherapy showing low response rate, and there was a lack of effective systemic therapies in the past. Due to the high risk (ca. 15-20%) of distant metastasis and limited systemic therapies, the 10-year survival rate of patients with advanced MTC was only 10-40% from the time of first metastasis. Over the past decade, targeted therapy for RET has developed rapidly, bringing hopes to patients with advanced and progressive MTC. Two multi-kinase inhibitors (MKIs) including Cabozantinib and Vandetanib have been shown to increase progression-free survival (PFS) for patients with metastatic MTC and have been approved as choices of first-line treatment. However, these MKIs have not prolonged overall survival (OS) and their utility is limited due to high rates of off-target toxicities. Recently, new generation TKIs, including Selpercatinib and Pralsetinib, have demonstrated highly selective efficacy against RET and more favorable side effect profiles, and gained approval as second-line treatment options. Despite the ongoing development of RET inhibitors, the management of advanced and progressive MTC remains challenging, drug resistance remains the main reason for treatment failure, and the mechanisms are still unclear. Besides, new promising therapeutic approaches, such as novel drug combinations and next generation RET inhibitors are under development. Herein, we overview the pathogenesis, molecular genetics and current management approaches of MTC, and focus on the recent advances of RET inhibitors, summarize the current situation and unmet needs of these RET inhibitors in MTC, and provide an overview of novel strategies for optimizing therapeutic effects.