Induction of the human CDC45 gene promoter activity by natural compound trans­resveratrol.

GGAA motifs in the human TP53 and HELB gene promoters play a part in responding to trans­resveratrol (Rsv) in HeLa S3 cells. This sequence is also present in the 5'­upstream region of the human CDC45 gene, which encodes a component of CMG DNA helicase protein complex. The cells were treated with Rsv (20 µM), then transcripts and the translated protein were analyzed by quantitative RT­PCR and western blotting, respectively. The results showed that the CDC45 gene and protein expression levels were induced after the treatment. To examine whether they were due to the activation of transcription, a 5'­upstream 556­bp of the CDC45 gene was cloned and inserted into a multi­cloning site of the Luciferase (Luc) expression vector. In the present study, various deletion/point mutation­introduced Luc expression plasmids were constructed and they were used for the transient transfection assay. The results showed that the GGAA motif, which is included in a putative RELB protein recognizing sequence, plays a part in the promoter activity with response to Rsv in HeLa S3 cells.

Generation of Connexin-Expressing Stable Cell Pools.

Stable cell pools have the advantage of providing a definite, consistent, and reproducible transmission of a transgene of interest, compared to transient expression from a plasmid transfection. Stably expressing a transgene of interest in cells under induction is a powerful way to (switch on and) study a gene function in both in vitro and in vivo assays. Taking advantage of the ability of lentivirus (LV) to promote transgene delivery, and genomic integration and expression in both dividing and nondividing cells, a doxycycline-inducible transfer vector expressing a bicistronic transgene was developed to study the function of connexins in HeLa DH cells. Here, delving on connexin 32 (Cx32), we report how to use the backbone of this vector as a tool to generate stable pools to study the function of a gene of interest (GOI), especially with assays involving Ca2+ imaging, employing the GCaMP6s indicator. We describe a step-by-step protocol to produce the LV particle by transient transfection and the direct use of the harvested LV stock to generate stable cell pools. We further present step-by-step immunolabeling protocols to characterize the transgene protein expression by confocal microscopy using an antibody that targets an extracellular domain epitope of Cx32 in living cells, and in fixed permeabilized cells using high affinity anti-Cx32 antibodies. Using common molecular biology laboratory techniques, this protocol can be adapted to generate stable pools expressing any transgene of interest, for both in vitro and in vivo functional assays, including molecular, immune, and optical assays.

Mannosylated polyethylenimine-cholesterol-based nanoparticles for targeted delivery of minicircle DNA vaccine against COVID-19 to antigen-presenting cells.

DNA vaccines can be a potential solution to protect global health, triggering both humoral and cellular immune responses. DNA vaccines are valuable in preventing intracellular pathogen infections, and therefore can be explored against coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2). This work explored different systems based on polyethylenimine (PEI), functionalized for the first time with both cholesterol (CHOL) and mannose (MAN) to deliver parental plasmid (PP) and minicircle DNA (mcDNA) vectors encoding the receptor-binding domain (RBD) of SARS-CoV-2 to antigen-presenting cells (APCs). For comparative purposes, three different systems were evaluated: PEI, PEI-CHOL and PEI-CHOL-MAN. The systems were prepared at various nitrogen-to-phosphate group (N/P) ratios and characterized in terms of encapsulation efficiency, surface charge, size, polydispersity index (PDI), morphology, and stability over time. Moreover, in vitro transfection studies of dendritic cells (JAWS II) and human fibroblast cells were performed. Viability studies assured the biocompatibility of all nanocarriers. Confocal microscopy studies confirmed intracellular localization of systems, resulting in enhanced cellular uptake using PEI-CHOL and PEI-CHOL-MAN systems when compared with the PEI system. Regarding the RBD expression, PEI-CHOL-MAN was the system that led to the highest levels of transcripts and protein expression in JAWS II cells. Furthermore, the nanosystems significantly stimulated pro-inflammatory cytokines production and dendritic cell maturation in vitro. Overall, mannosylated systems can be considered a valuable tool in the delivery of plasmid DNA or mcDNA vaccines to APCs.

Transfection of hypoxia-inducible factor-1α mRNA upregulates the expression of genes encoding angiogenic growth factors.

Hypoxia-Inducible Factor-1α (HIF-1α) has presented a new direction for ischemic preconditioning of surgical flaps to promote their survival. In a previous study, we demonstrated the effectiveness of HIF-1a DNA plasmids in this application. In this study, to avoid complications associated with plasmid use, we sought to express HIF-1α through mRNA transfection and determine its biological activity by measuring the upregulation of downstream angiogenic genes. We transfected six different HIF-1a mRNAs-one predominant, three variant, and two novel mutant isoforms-into primary human dermal fibroblasts using Lipofectamine, and assessed mRNA levels using RT-qPCR. At all time points examined after transfection (3, 6, and 10 h), the levels of HIF-1α transcript were significantly higher in all HIF-1α transfected cells relative to the control (all p < 0.05, unpaired Student's T-test). Importantly, the expression of HIF-1α transcription response genes (VEGF, ANG-1, PGF, FLT1, and EDN1) was significantly higher in the cells transfected with all isoforms than with the control at six and/or ten hours post-transfection. All isoforms were transfected successfully into human fibroblast cells, resulting in the rapid upregulation of all five downstream angiogenic targets tested. These findings support the potential use of HIF-1α mRNA for protecting ischemic dermal flaps.

PEI-based functional materials: Fabrication techniques, properties, and biomedical applications.

Cationic polymers have recently attracted considerable interest as research breakthroughs for various industrial and biomedical applications. They are particularly interesting due to their highly positive charges, acceptable physicochemical properties, and ability to undergo further modifications, making them attractive candidates for biomedical applications. Polyethyleneimines (PEIs), as the most extensively utilized polymers, are one of the valuable and prominent classes of polycations. Owing to their flexible polymeric chains, broad molecular weight (MW) distribution, and repetitive structural units, their customization for functional composites is more feasible. The specific beneficial attributes of PEIs could be introduced by purposeful functionalization or modification, long service life, biocompatibility, and distinct geometry. Therefore, PEIs have significant potential in biotechnology, medicine, and bioscience. In this review, we present the advances in PEI-based nanomaterials, their transfection efficiency, and their toxicity over the past few years. Furthermore, the potential and suitability of PEIs for various applications are highlighted and discussed in detail. This review aims to inspire readers to investigate innovative approaches for the design and development of next-generation PEI-based nanomaterials possessing cutting-edge functionalities and appealing characteristics.

Characterization of Skeletal Muscle Regeneration Revealed a Novel Growth Network Induced by Molecular Acupuncture-like Transfection.

The low efficiency of in vivo transfection of a few fibres revealed a novel tissue network that temporally amplified growth stimulation in the entire regenerating rat soleus muscle. This acupuncture-like effect was demonstrated when the fibres began to grow after complete fibre degradation, synchronous inflammation, myoblast and myotube formation. Neonatal sarcoplasmic/endoplasmic reticulum ATPase (SERCA1b) was first detected in this system. The neonatal, fast and slow SERCA isoforms displayed consequent changes with innervation and differentiation, recapitulating events in muscle development. In vivo transfection of myotubes with plasmids expressing dominant negative Ras or a calcineurin inhibitor peptide (Cain/cabin) proved that expression of the slow myosin heavy chain and the slow muscle type SERCA2a are differentially regulated. In vivo transfection of a few nuclei of myotubes with dnRas or SERCA1b shRNA stimulated fibre size growth in the whole regenerating muscle but only until the full size had been reached. Growth stimulation by Ras and SERCA1b antisense was abolished by co-transfection of Cain or with perimuscular injection of IL4 antibody. This revealed a novel signalling network resembling scale-free networks which, starting from transfected fibre myonuclei as "hubs", can amplify growth stimulation uniformly in the entire regenerating muscle.

Enabling High-throughput Transgene Expression Studies Using Automated Liquid Handling for Etiolated Maize Leaf Protoplasts.

The field of plant biotechnology has witnessed remarkable advancements in recent years, revolutionizing the ability to manipulate and engineer plants for various purposes. However, as research in this field increases in diversity and becomes increasingly sophisticated, the need for early, efficient, dependable, and high-throughput transient screening solutions to narrow down strategies proceeding to stable transformation is more apparent. One method that has re-emerged in recent years is the utilization of plant protoplast, for which methods of isolation and transfection are available in numerous species, tissues, and developmental stages. This work describes a simple automated protocol for the randomized preparation of plasmid within a 96-well plate, a method for the isolation of etiolated maize leaf protoplast, and an automated transfection procedure. The adoption of automated solutions in plant biotechnology, exemplified by these novel liquid handling protocols for plant protoplast transfection, represents a significant advancement over manual methods. By leveraging automation, researchers can easily overcome the limitations of traditional methods, enhance efficiency, and accelerate scientific progress.

Flow Cytometry Quantification of Transient Transfections in Mammalian Cells.

Flow cytometry is a powerful quantitative assay supporting high-throughput collection of single-cell data with a high dynamic range. For flow cytometry to yield reproducible data with a quantitative relationship to the underlying biology, however, requires that (1) appropriate process controls are collected along with experimental samples, (2) these process controls are used for unit calibration and quality control, and (3) data are analyzed using appropriate statistics. To this end, this chapter describes methods for quantitative flow cytometry through the addition of process controls and analyses, thereby enabling better development, modeling, and debugging of engineered biological organisms. The methods described here have specifically been developed in the context of transient transfections in mammalian cells but may in many cases be adaptable to other categories of transfection and other types of cells.

High-Throughput Spectroscopic Analysis of mRNA Capping Level.

Eukaryotic mRNAs are characterized by terminal 5' cap structures and 3' polyadenylation sites, which are essential for posttranscriptional processing, translation initiation, and stability. Here, we describe a novel biosensor method designed to detect the presence of both cap structures and polyadenylation sites on mRNA molecules. This novel biosensor is sensitive to mRNA degradation and can quantitatively determine capping levels of mRNA molecules within a mixture of capped and uncapped mRNA molecules. The biosensor displays a constant dynamic range between 254 nt and 6507 nt with reproducible sensitivity to increases in capping level of at least 20% and a limit of detection of 2.4 pmol of mRNA. Overall, the biosensor can provide key information about mRNA quality before mammalian cell transfection.

Impact of Linker Groups on Self-Assembly, Gene Transfection, Antibacterial Activity, and In Vitro Cytotoxicity of Cationic Bolaamphiphiles.

Cationic bolaamphiphiles have gained significant attention in various research fields, including materials science, drug delivery, and gene therapy, due to their unique properties and potential applications. The objective of the current research is to develop more effective cationic bolaamphiphiles. Thus, we have designed and synthesized two cationic bolaamphiphiles (-(CH2)12(2,3-dihydroxy-N,N-dimethyl-N-(3-ureidopropyl)propan-1-aminium chloride))2 (C12(DDUPPAC)2)) and (-(CH2)12(N-(3-(carbamoyloxy)propyl)-2,3-dihydroxy-N,N-dimethylpropan-1-aminium chloride)2 (C12(CPDDPAC)2) containing urea and urethane linkages, respectively. We have investigated their self-assembly properties in water using several techniques, including surface tension, electrical conductivity, fluorescence probe, calorimetry, dynamic light scattering, and atomic force microscopy. Their biological applications, e.g., in vitro gene transfection, antibacterial activity, and cytotoxicity, were studied. Both bolaamphiphiles were observed to produce aggregates larger than spherical micelles above a relatively low critical aggregation concentration (cac). The calorimetric experiments suggested the thermodynamically favorable spontaneous aggregation of both bolaforms in water. The results of interaction studies led to the conclusion that C12(CPDDPAC)2 binds DNA with a greater affinity than C12(DDUPPAC)2. Also, C12(CPDDPAC)2 is found to act as a more efficient gene transfection vector than C12(DDUPPAC)2 in 264.7 cell lines. The in vitro cytotoxicity assay using MTT, however, revealed that neither of the bolaamphiphiles was toxic, even at higher quantities. Additionally, both bolaforms show beneficial antibacterial activity.

GalNAc- or Mannose-PEG-Functionalized Polyplexes Enable Effective Lectin-Mediated DNA Delivery.

A cationic, dendrimer-like oligo(aminoamide) carrier with four-arm topology based on succinoyl tetraethylene pentamine and histidines, cysteines, and N-terminal azido-lysines was screened for plasmid DNA delivery on various cell lines. The incorporated azides allow modification with various shielding agents of different polyethylene glycol (PEG) lengths and/or different ligands by copper-free click reaction, either before or after polyplex formation. Prefunctionalization was found to be advantageous over postfunctionalization in terms of nanoparticle formation, stability, and efficacy. A length of 24 ethylene oxide repetition units and prefunctionalization of ≥50% of azides per carrier promoted optimal polyplex shielding. PEG shielding resulted in drastically reduced DNA transfer, which could be successfully restored by active lectin targeting via novel GalNAc or mannose ligands, enabling enhanced receptor-mediated endocytosis of the carrier system. The involvement of the asialoglycoprotein receptor (ASGPR) in the uptake of GalNAc-functionalized polyplexes was confirmed in the ASGPR-positive hepatocarcinoma cell lines HepG2 and Huh7. Mannose-modified polyplexes showed superior cellular uptake and transfection efficacy compared to unmodified and shielded polyplexes in mannose-receptor-expressing dendritic cell-like DC2.4 cells.

Nontoxic, Biodegradable Hyperbranched Poly(ß-amino ester)s for Efficient siRNA Delivery and Gene Silencing.

RNA interference (RNAi)-mediated gene silencing is a promising therapeutic approach to treat various diseases, but safe and efficient delivery remains a major challenge to its clinical application. Non-viral gene vectors, such as poly(ß-amino esters) (pBAEs), have emerged as a potential candidate due to their biodegradability, low toxicity profile, ease of synthesis, and high gene transfection efficiency for both DNA and siRNA delivery. However, achieving significant gene silencing using pBAEs often requires a large amount of polymer carrier (with polymer/siRNA weight ratio >100) or high siRNA dose (>100 nM), which might potentially exacerbate toxicity concerns during delivery. To overcome these barriers, we designed and optimized a series of hyperbranched pBAEs capable of efficiently condensing siRNA and achieving excellent silencing efficiency at a lower polymer/siRNA weight ratio (w/w) and siRNA dose. Through modulation of monomer combinations and branching density, we identified the top-performing hyperbranched pBAEs, named as h(A2B3)-1, which possess good siRNA condensation ability, low cytotoxicity, and high cellular uptake efficiency. Compared with Lipofectamine 2000, h(A2B3)-1 achieved lower cytotoxicity and higher siRNA silencing efficiency in HeLa cells at a polymer/siRNA weight ratio of 30 and 30 nM siRNA dose. Notably, h(A2B3)-1 enhanced the gene uptake in primary neural cells and effectively silenced the target gene in hard-to-transfect primary cortical neurons and oligodendrocyte progenitor cells, with gene knockdown efficiencies of 34.8 and 53.4% respectively. By incorporating a bioreducible disulfide compartment into the polymer backbone, the cytocompatibility of the h(A2B3)-1 was greatly enhanced while maintaining their good transfection efficiency. Together, the low cytotoxicity and high siRNA transfection efficiency of hyperbranched h(A2B3)-1 in this study demonstrated their great potential as a non-viral gene vector for efficient siRNA delivery and RNAi-mediated gene silencing. This provides valuable insight into the future development of safe and efficient non-viral siRNA delivery systems as well as their translation into clinical applications.

Electrostatic adsorption of polyanions onto lipid nanoparticles controls uptake, trafficking, and transfection of RNA and DNA therapies.

Rapid advances in nucleic acid therapies highlight the immense therapeutic potential of genetic therapeutics. Lipid nanoparticles (LNPs) are highly potent nonviral transfection agents that can encapsulate and deliver various nucleic acid therapeutics, including but not limited to messenger RNA (mRNA), silencing RNA (siRNA), and plasmid DNA (pDNA). However, a major challenge of targeted LNP-mediated systemic delivery is the nanoparticles' nonspecific uptake by the liver and the mononuclear phagocytic system, due partly to the adsorption of endogenous serum proteins onto LNP surfaces. Tunable LNP surface chemistries may enable efficacious delivery across a range of organs and cell types. Here, we describe a method to electrostatically adsorb bioactive polyelectrolytes onto LNPs to create layered LNPs (LLNPs). LNP cores varying in nucleic acid cargo and component lipids were stably layered with four biologically relevant polyanions: hyaluronate (HA), poly-L-aspartate (PLD), poly-L-glutamate (PLE), and polyacrylate (PAA). We further investigated the impact of the four surface polyanions on the transfection and uptake of mRNA- and pDNA-loaded LNPs in cell cultures. PLD- and PLE-LLNPs increased mRNA transfection twofold over unlayered LNPs in immune cells. HA-LLNPs increased pDNA transfection rates by more than twofold in epithelial and immune cells. In a healthy C57BL/6 murine model, PLE- and HA-LLNPs increased transfection by 1.8-fold to 2.5-fold over unlayered LNPs in the liver and spleen. These results suggest that LbL assembly is a generalizable, highly tunable platform to modify the targeting specificity, stability, and transfection efficacy of LNPs, as well as incorporate other charged targeting and therapeutic molecules into these systems.

Antibiotic-Free Gene Vectors: A 25-Year Journey to Clinical Trials.

Until very recently, the major use, for gene therapy, specifically of linear or circular DNA, such as plasmids, was as ancillary products for viral vectors' production or as a genetic template for mRNA production. Thanks to targeted and more efficient physical or chemical delivery techniques and to the refinement of their structure, non-viral plasmid DNA are now under intensive consideration as pharmaceutical drugs. Plasmids traditionally carry an antibiotic resistance gene for providing the selection pressure necessary for maintenance in a bacterial host. Nearly a dozen different antibiotic-free gene vectors have now been developed and are currently assessed in preclinical assays and phase I/II clinical trials. Their reduced size leads to increased transfection efficiency and prolonged transgene expression. In addition, associating non-viral gene vectors and DNA transposons, which mediate transgene integration into the host genome, circumvents plasmid dilution in dividing eukaryotic cells which generate a loss of the therapeutic gene. Combining these novel molecular tools allowed a significantly higher yield of genetically engineered T and Natural Killer cells for adoptive immunotherapies due to a reduced cytotoxicity and increased transposition rate. This review describes the main progresses accomplished for safer, more efficient and cost-effective gene and cell therapies using non-viral approaches and antibiotic-free gene vectors.

Safety and Efficacy of IL-12 Plasmid DNA Transfection into Pig Skin: Supportive Data for Human Clinical Trials on Gene Therapy and Vaccination.

Gene electrotransfer (GET) of plasmids encoding interleukin 12 (IL-12) has already been used for the treatment of various types of tumors in human oncology and as an adjuvant in DNA vaccines. In recent years, we have developed a plasmid encoding human IL-12 (phIL12) that is currently in a phase I clinical study. The aim was to confirm the results of a non-clinical study in mice on pharmacokinetic characteristics and safety in a porcine model that better resembled human skin. The GET of phIL12 in the skin was performed on nine pigs using different concentrations of plasmid phIL12 and invasive (needle) or noninvasive (plate) types of electrodes. The results of our study demonstrate that the GET of phIL-12 with needle electrodes induced the highest expression of IL-12 at the protein level on day 7 after the procedure. The plasmid was distributed to all tested organs; however, its amount decreased over time and was at a minimum 28 days after GET. Based on plasmid copy number and expression results, together with blood analysis, we showed that IL-12 GET is safe in a porcine animal model. Furthermore, we demonstrated that pigs are a valuable model for human gene therapy safety studies.

A High-Throughput Microdroplet-Based Single Cell Transfection Method for Gene Knockout Based on the CRISPR/Cas9 System.

The efficient application of the newly developed gene-editing method CRISPR/Cas9 requires more accurate intracellular gene delivery. Traditional delivery approaches, such as lipotransfection and non-viral delivery methods, must contend with major problems to overcome the drawbacks of low efficiency, high toxicity, and cell-type dependency. The high-throughput microdroplet-based single-cell transfection method presented herein provides an alternative method for delivering genome-editing reagents into single living cells. By accurately controlling the number of exogenous plasmids in microdroplets, this method can achieve high-efficiency delivery of nucleic acids to different types of single cells. This paper presents a high-throughput quantitative DNA transfection method for single cells and explores the optimal DNA transfection conditions for specific cell lines. The transfection efficiency of cells at different concentrations of DNA in microdroplets is measured. Under the optimized transfection conditions, the method is used to construct gene-knockout cancer cell lines to determine specific gene functions through the CRISPR/Cas9 knockout system. In a case study, the migration ability of TRIM72 knockout cancer cells is inhibited, and the tumorigenicity of cells in a zebrafish tumor model is reduced. A single-cell microfluidic chip is designed to achieve CRISPR/Cas9 DNA transfection, dramatically improving the transfection efficiency of difficult-to-transfect cells. This research demonstrates that the microdroplet method developed herein has a unique advantage in CRISPR/Cas9 gene-editing applications.

Coupling of nanostraws with diverse physicochemical perforation strategies for intracellular DNA delivery.

Effective intracellular DNA transfection is imperative for cell-based therapy and gene therapy. Conventional gene transfection methods, including biochemical carriers, physical electroporation and microinjection, face challenges such as cell type dependency, low efficiency, safety concerns, and technical complexity. Nanoneedle arrays have emerged as a promising avenue for improving cellular nucleic acid delivery through direct penetration of the cell membrane, bypassing endocytosis and endosome escape processes. Nanostraws (NS), characterized by their hollow tubular structure, offer the advantage of flexible solution delivery compared to solid nanoneedles. However, NS struggle to stably self-penetrate the cell membrane, resulting in limited delivery efficiency. Coupling with extra physiochemical perforation strategies is a viable approach to improve their performance. This study systematically compared the efficiency of NS coupled with polyethylenimine (PEI) chemical modification, mechanical force, photothermal effect, and electric field on cell membrane perforation and DNA transfection. The results indicate that coupling NS with PEI modification, mechanical force, photothermal effects provide limited enhancement effects. In contrast, NS-electric field coupling significantly improves intracellular DNA transfection efficiency. This work demonstrates that NS serve as a versatile platform capable of integrating various physicochemical strategies, while electric field coupling stands out as a form worthy of primary consideration for efficient DNA transfection.

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.

Hybrid Lipid Nanocapsules: A Robust Platform for mRNA Delivery.

The success of the mRNA vaccine against COVID-19 has garnered significant interest in the development of mRNA therapeutics against other diseases, but there remains a strong need for a stable and versatile delivery platform for these therapeutics. In this study, we report on a family of robust hybrid lipid nanocapsules (hLNCs) for the delivery of mRNA. The hLNCs are composed of kolliphore HS15, labrafac lipophile WL1349, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and a conjugate of oleic acid (OA) and polyethylenimines of varying size (PEI─0.8, 1.8, and 25 kDa). They are prepared by a solvent-free, temperature-phase inversion method, yielding an average size of ∼40 nm and a particle distribution index (PDI) < 0.2. We demonstrate that the PDI remains <0.2 over a wide pH range and in a wide range of medium. We further show that the PDI and the functionality of mRNA condensed on the particles are robust to drying in a sugar glass and subsequent rehydration. Finally, we demonstrate that mRNA-loaded hLNCs yield reasonable transfection in vitro and in vivo settings.

Imaging and Assay of the Dynamics of Cytotoxic Huntingtin (HTT) Protein Aggregates Regulated by lncRNAs.

Huntington's disease (HD) pathogenesis involves deregulation of coding and noncoding RNA transcripts of which the involvement of long noncoding RNAs (lncRNA) has been realized recently. Of these, Meg3, Neat1, and Xist showed a consistent and significant increase in HD cell and animal models. In the present study, we formulate a methodology to visualize and quantify intracellular aggregates formed by mutant HTT protein. This method employs the use of both confocal laser scanning and super resolution (N-SIM) microscopy to accurately estimate aggregate numbers. Further, to determine the role of two lncRNAs Meg3 and Neat1 in the formation of aggregates of mutant HTT, we used commercially available siRNAs against Meg3 and Neat1 for transiently knocking them down in mouse Neuro2a and human SHSY5Y cells. Co-transfection of 83Q-DsRed and siRNA specific for Neat1 or Meg3 resulted in decreased intracellular aggregates of 83Q-DsRed in both the cell lines. We have established a quantitative method to estimate and directly or indirectly modulate the formation of mutant HTT aggregates.