Comparative Analysis of the Physicochemical and Biological Characteristics of Freeze-Dried PEGylated Cationic Solid Lipid Nanoparticles.

Cationic solid-lipid nanoparticles (cSLNs) have become a promising tool for gene and RNA therapies. PEGylation (PEG) is crucial in enhancing particle stability and protection. We evaluated the impact of PEG on the physicochemical and biological characteristics of cholesteryl-oleate cSLNs (CO-cSLNs). Several parameters were analyzed, including the particle size, polydispersity index, zeta potential, shape, stability, cytotoxicity, and loading efficiency. Five different formulations with specific PEGs were developed and compared in both suspended and freeze-dried states. Small, homogeneous, and cationic suspended nanoparticles were obtained, with the Gelucire 50/13 (PEG-32 hydrogenated palm glycerides; Gelucire) and DSPE-mPEG2000 (1,2-distearoyl-phosphatidylethanolamine-methyl-polyethyleneglycol conjungate-2000; DSPE) formulations exhibiting the smallest particle size (~170 nm). Monodisperse populations of freeze-dried nanoparticles were also achieved, with particle sizes ranging from 200 to 300 nm and Z potential values of 30-35 mV. Notably, Gelucire again produced the smallest particle size (211.1 ± 22.4), while the DSPE and Myrj S100 (polyoxyethylene (100) stearate; PEG-100 Stearate) formulations had similar particle sizes to CO-cSLNs (~235 nm). The obtained PEGylated nanoparticles showed suitable properties: they were nontoxic, had acceptable morphology, were capable of forming SLNplexes, and were stable in both suspended and lyophilized states. These PEG-cSLNs are a potential resource for in vivo assays and have the advantage of employing cost-effective PEGs. Optimizing the lyophilization process and standardizing parameters are also recommended to maintain nanoparticle integrity.

Interleukin-7 receptor signaling is crucial for enhancer-dependent TCRδ germline transcription mediated through STAT5 recruitment.

γδ T cells play important roles in immune responses by rapidly producing large quantities of cytokines. Recently, γδ T cells have been found to be involved in tissue homeostatic regulation, playing roles in thermogenesis, bone regeneration and synaptic plasticity. Nonetheless, the mechanisms involved in γδ T-cell development, especially the regulation of TCRδ gene transcription, have not yet been clarified. Previous studies have established that NOTCH1 signaling plays an important role in the Tcrg and Tcrd germline transcriptional regulation induced by enhancer activation, which is mediated through the recruitment of RUNX1 and MYB. In addition, interleukin-7 signaling has been shown to be required for Tcrg germline transcription, VγJγ rearrangement and γδ T-lymphocyte generation as well as for promoting T-cell survival. In this study, we discovered that interleukin-7 is required for the activation of enhancer-dependent Tcrd germline transcription during thymocyte development. These results indicate that the activation of both Tcrg and Tcrd enhancers during γδ T-cell development in the thymus depends on the same NOTCH1- and interleukin-7-mediated signaling pathways. Understanding the regulation of the Tcrd enhancer during thymocyte development might lead to a better understanding of the enhancer-dependent mechanisms involved in the genomic instability and chromosomal translocations that cause leukemia.

Differently Regulated Gene-Specific Activity of Enhancers Located at the Boundary of Subtopologically Associated Domains: TCRα Enhancer.

Enhancers activate transcription through long-distance interactions with their cognate promoters within a particular subtopologically associated domain (sub-TAD). The TCRα enhancer (Eα) is located at the sub-TAD boundary between the TCRα and DAD1 genes and regulates transcription toward both sides in an ∼1-Mb region. Analysis of Eα activity in transcribing the unrearranged TCRα gene at the 5'-sub-TAD has defined Eα as inactive in CD4-CD8- thymocytes, active in CD4+CD8+ thymocytes, and strongly downregulated in CD4+ and CD8+ thymocytes and αß T lymphocytes. Despite its strongly reduced activity, Eα is still required for high TCRα transcription and expression of TCRαß in mouse and human T lymphocytes, requiring collaboration with distant sequences for such functions. Because VαJα rearrangements in T lymphocytes do not induce novel long-range interactions between Eα and other genomic regions that remain in cis after recombination, strong Eα connectivity with the 3'-sub-TAD might prevent reduced transcription of the rearranged TCRα gene. Our analyses of transcriptional enhancer dependence during T cell development and non-T lineage tissues at the 3'-sub-TAD revealed that Eα can activate the transcription of specific genes, even when it is inactive to transcribe the TCRα gene at the 5'-sub-TAD. Hence distinct requirements for Eα function are necessary at specific genes at both sub-TADs, implying that enhancers do not merely function as chromatin loop anchors that nucleate the formation of factor condensates to increase gene transcription initiated at their cognate promoters. The observed different regulated Eα activity for activating specific genes at its flanking sub-TADs may be a general feature for enhancers located at sub-TAD boundaries.

Regulation of T-cell Receptor Gene Expression by Three-Dimensional Locus Conformation and Enhancer Function.

The adaptive immune response in vertebrates depends on the expression of antigen-specific receptors in lymphocytes. T-cell receptor (TCR) gene expression is exquisitely regulated during thymocyte development to drive the generation of αß and γδ T lymphocytes. The TCRα, TCRß, TCRγ, and TCRδ genes exist in two different configurations, unrearranged and rearranged. A correctly rearranged configuration is required for expression of a functional TCR chain. TCRs can take the form of one of three possible heterodimers, pre-TCR, TCRαß, or TCRγδ which drive thymocyte maturation into αß or γδ T lymphocytes. To pass from an unrearranged to a rearranged configuration, global and local three dimensional (3D) chromatin changes must occur during thymocyte development to regulate gene segment accessibility for V(D)J recombination. During this process, enhancers play a critical role by modifying the chromatin conformation and triggering noncoding germline transcription that promotes the recruitment of the recombination machinery. The different signaling that thymocytes receive during their development controls enhancer activity. Here, we summarize the dynamics of long-distance interactions established through chromatin regulatory elements that drive transcription and V(D)J recombination and how different signaling pathways are orchestrated to regulate the activity of enhancers to precisely control TCR gene expression during T-cell maturation.

Innovative Therapeutic and Delivery Approaches Using Nanotechnology to Correct Splicing Defects Underlying Disease.

Alternative splicing of pre-mRNA contributes strongly to the diversity of cell- and tissue-specific protein expression patterns. Global transcriptome analyses have suggested that >90% of human multiexon genes are alternatively spliced. Alterations in the splicing process cause missplicing events that lead to genetic diseases and pathologies, including various neurological disorders, cancers, and muscular dystrophies. In recent decades, research has helped to elucidate the mechanisms regulating alternative splicing and, in some cases, to reveal how dysregulation of these mechanisms leads to disease. The resulting knowledge has enabled the design of novel therapeutic strategies for correction of splicing-derived pathologies. In this review, we focus primarily on therapeutic approaches targeting splicing, and we highlight nanotechnology-based gene delivery applications that address the challenges and barriers facing nucleic acid-based therapeutics.

Drosophila Prp40 localizes to the histone locus body and regulates gene transcription and development.

In eukaryotes, a large amount of histones need to be synthesized during the S phase of the cell cycle to package newly synthesized DNA into chromatin. The transcription and 3' end processing of histone pre-mRNAs are controlled by the histone locus body (HLB), which is assembled on the shared promoter for H3 and H4 Here, we identified the Drosophila Prp40 pre-mRNA processing factor (dPrp40, annotated as CG3542) as a novel HLB component. We showed that dPrp40 is essential for Drosophila development, with functionally conserved activity in vertebrates and invertebrates. We observed that dPrp40 is fundamental in endocycling cells, highlighting a role for this factor in mediating replication efficiency in vivo The depletion of dPrp40 from fly cells inhibited the transcription, but not the 3' end processing, of histone mRNA in a H3- and H4-promoter-dependent manner. Our results establish that dPrp40 is an essential protein for Drosophila development that can localize to the HLB and might participate in histone mRNA biosynthesis.

Role for the splicing factor TCERG1 in Cajal body integrity and snRNP assembly.

Cajal bodies are nuclear organelles involved in the nuclear phase of small nuclear ribonucleoprotein (snRNP) biogenesis. In this study, we identified the splicing factor TCERG1 as a coilin-associated factor that is essential for Cajal body integrity. Knockdown of TCERG1 disrupts the localization of the components of Cajal bodies, including coilin and NOLC1, with coilin being dispersed in the nucleoplasm into numerous small foci, without affecting speckles, gems or the histone locus body. Furthermore, the depletion of TCERG1 affects the recruitment of Sm proteins to uridine-rich small nuclear RNAs (snRNAs) to form the mature core snRNP. Taken together, the results of this study suggest that TCERG1 plays an important role in Cajal body formation and snRNP biogenesis.

Development and characterization of an improved formulation of cholesteryl oleate-loaded cationic solid-lipid nanoparticles as an efficient non-viral gene delivery system.

Nanoparticle-mediated plasmid delivery is considered a useful tool to introduce foreign DNA into the cells for the purpose of DNA vaccination and/or gene therapy. Cationic solid-lipid nanoparticles (cSLNs) are considered one of the most promising non-viral vectors for nucleic acid delivery. Based on the idea that the optimization of the components is required to improve transfection efficiency, the present study aimed to formulate and characterize cholesteryl oleate-containing solid-lipid nanoparticles (CO-SLNs) incorporating protamine (P) to condense DNA to produce P:DNA:CO-SLN complexes as non-viral vectors for gene delivery with reduced cytotoxicity and high cellular uptake efficiency. For this purpose, CO-SLNs were used to prepare DNA complexes with and without protamine as DNA condenser and nuclear transfer enhancer. The main physicochemical characteristics, binding capabilities, cytotoxicity and cellular uptake of these novel CO-SLNs were analyzed. Positively charged spherical P:DNA:CO-SLN complexes with a particle size ranging from 330.1 ± 14.8 nm to 347.0 ± 18.5 nm were obtained. Positive results were obtained in the DNase I protection assay with a protective effect of the genetic material and 100% loading efficiency was achieved at a P:DNA:CO-SLN ratio of 2:1:7. Transfection studies in human embryonic kidney (HEK293T) cells showed the versatility of adding protamine to efficiently transfect cells, widening the potential applications of CO-SLN-based vectors, since the incorporation of protamine induced almost a 200-fold increase in the transfection capacity of CO-SLNs without toxicity. These results indicate that CO-SLNs with protamine are a safe and effective platform for non-viral nucleic acid delivery.

Improved synthesis and characterization of cholesteryl oleate-loaded cationic solid lipid nanoparticles with high transfection efficiency for gene therapy applications.

The development of new nanoparticle formulations that are capable of high transfection efficiency without toxicity is essential to provide new tools for gene therapy. However, the issues of complex, poorly reproducible manufacturing methods, and low efficiencies during in vivo testing have prevented translation to the clinic. We have previously reported the use of cholesteryl oleate as a novel excipient for solid lipid nanoparticles (SLNs) for the development of highly efficient and nontoxic nucleic acid delivery carriers. Here, we performed an extensive characterization of this novel formulation to make the scale up under Good Manufacturing Practice (GMP) possible. We also describe the complete physicochemical and biological characterization of cholesteryl oleate-loaded SLNs to ensure the reproducibility of this formula and the preservation of its characteristics before and after the lyophilization process. We defined the best manufacturing method and studied the influence of some parameters on the obtained nanoparticles using the Quality by Design (ICH Q8) guideline to obtain cholesteryl oleate-loaded SLNs that remain stable during storage and guarantee in vitro nucleic acid delivery efficacy. Our results indicate that this improved formulation is suitable for gene therapy with the possibility of scale-up the manufacturing of nanoparticles under GMP conditions.

Notch Signaling Controls Transcription via the Recruitment of RUNX1 and MYB to Enhancers during T Cell Development.

Tcrd and Tcrg display identical developmental programs that depend on the activity of the enhancers Eδ and Eγ being "on" in pre-ß-selection thymocytes to activate transcription and V(D)J recombination of the unrearranged genes and "off" in post-ß-selection CD4+CD8+ double-positive thymocytes to inhibit transcription of the rearranged genes and avoid the expression of TCR δ- and TCR γ-chains in αß T lymphocytes. Eδ and Eγ activity depends on transcription factor binding to essential Runx and Myb sites and parallels that of Notch signaling. We performed Notch gain- and loss-of-function experiments and found that Notch signaling activates Tcrd and Tcrg transcription by favoring the recruitment of RUNX1 and MYB to the enhancers. Our results suggest that the dissociation of RUNX1 and MYB from Eδ and Eγ chromatin in double-positive thymocytes, which results in enhancer inactivation, is caused by decreased Notch signaling triggered by pre-TCR signaling, thereby deciphering the molecular mechanism of Tcrd and Tcrg silencing during ß-selection. These findings reveal a novel molecular mechanism for gene regulation via Notch signaling through the recruitment of RUNX1 and MYB to enhancer chromatin during thymocyte development.

Cholesteryl oleate-loaded cationic solid lipid nanoparticles as carriers for efficient gene-silencing therapy.

BACKGROUND: Cationic solid lipid nanoparticles (SLNs) have been given considerable attention for therapeutic nucleic acid delivery owing to their advantages over viral and other nanoparticle delivery systems. However, poor delivery efficiency and complex formulations hinder the clinical translation of SLNs. AIM: The aim of this study was to formulate and characterize SLNs incorporating the cholesterol derivative cholesteryl oleate to produce SLN-nucleic acid complexes with reduced cytotoxicity and more efficient cellular uptake. METHODS: Five cholesteryl oleate-containing formulations were prepared. Laser diffraction and laser Doppler microelectrophoresis were used to evaluate particle size and zeta potential, respectively. Nanoparticle morphology was analyzed using electron microscopy. Cytotoxicity and cellular uptake of lipoplexes were evaluated using flow cytometry and fluorescence microscopy. The gene inhibition capacity of the lipoplexes was assessed using siRNAs to block constitutive luciferase expression. RESULTS: We obtained nanoparticles with a mean diameter of approximately 150-200 nm in size and zeta potential values of 25-40 mV. SLN formulations with intermediate concentrations of cholesteryl oleate exhibited good stability and spherical structures with no aggregation. No cell toxicity of any reference SLN was observed. Finally, cellular uptake experiments with DNA-and RNA-SLNs were performed to select one reference with superior transient transfection efficiency that significantly decreased gene activity upon siRNA complexation. CONCLUSION: The results indicate that cholesteryl oleate-loaded SLNs are a safe and effective platform for nonviral nucleic acid delivery.

Targeting proteins to RNA transcription and processing sites within the nucleus.

Studies of the spatial organization of the highly compartmentalized eukaryotic nucleus and dynamics of transcription and RNA processing within it are fundamental for fully understanding how gene expression is regulated in the cell. Although some progress has been made in deciphering the functional consequences of this complex network of interacting molecules in the context of nuclear organization, how proteins and RNA move in the nucleus and how the transcription and RNA processing machineries find their targets are important questions that remain largely unexplored. Here, we review major hallmarks and novel insights regarding the movement of RNA and proteins in the context of nuclear organization as well as the mechanisms by which the proteins involved in RNA processing localize to specific nuclear compartments.

Targeting Splicing in the Treatment of Human Disease.

The tightly regulated process of precursor messenger RNA (pre-mRNA) alternative splicing (AS) is a key mechanism in the regulation of gene expression. Defects in this regulatory process affect cellular functions and are the cause of many human diseases. Recent advances in our understanding of splicing regulation have led to the development of new tools for manipulating splicing for therapeutic purposes. Several tools, including antisense oligonucleotides and trans-splicing, have been developed to target and alter splicing to correct misregulated gene expression or to modulate transcript isoform levels. At present, deregulated AS is recognized as an important area for therapeutic intervention. Here, we summarize the major hallmarks of the splicing process, the clinical implications that arise from alterations in this process, and the current tools that can be used to deliver, target, and correct deficiencies of this key pre-mRNA processing event.

Improved formulation of cationic solid lipid nanoparticles displays cellular uptake and biological activity of nucleic acids.

Non-viral delivery using cationic solid lipid nanoparticles (SLNs) represents a useful strategy to introduce large DNA and RNA molecules to target cells. A careful selection of components and their amounts is critical to improve transfection efficiency. In this work, a selected and optimized formulation of SLNs was used to efficiently transfect circular DNA and linear RNA molecules into cells. We characterized the main physicochemical characteristics and binding capabilities of these SLNs and show that they deliver DNA and RNA molecules into cells where they display full bioactivity at nontoxic concentrations using fluorescence- and luminescence-based methodologies. Hence, we established a novel and simple SLN formulation as a powerful tool for future therapeutic use.

Transcriptional Elongation Regulator 1 Affects Transcription and Splicing of Genes Associated with Cellular Morphology and Cytoskeleton Dynamics and Is Required for Neurite Outgrowth in Neuroblastoma Cells and Primary Neuronal Cultures.

TCERG1 is a highly conserved human protein implicated in interactions with the transcriptional and splicing machinery that is associated with neurodegenerative disorders. Biochemical, neuropathological, and genetic evidence suggests an important role for TCERG1 in Huntington's disease (HD) pathogenesis. At present, the molecular mechanism underlying TCERG1-mediated neuronal effects is unknown. Here, we show that TCERG1 depletion led to widespread alterations in mRNA processing that affected different types of alternative transcriptional or splicing events, indicating that TCERG1 plays a broad role in the regulation of alternative splicing. We observed considerable changes in the transcription and alternative splicing patterns of genes involved in cytoskeleton dynamics and neurite outgrowth. Accordingly, TCERG1 depletion in the neuroblastoma SH-SY5Y cell line and primary mouse neurons affected morphogenesis and resulted in reduced dendritic outgrowth, with a major effect on dendrite ramification and branching complexity. These defects could be rescued by ectopic expression of TCERG1. Our results indicate that TCERG1 affects expression of multiple mRNAs involved in neuron projection development, whose misregulation may be involved in TCERG1-linked neurological disorders.

The in vivo dynamics of TCERG1, a factor that couples transcriptional elongation with splicing.

Coupling between transcription and RNA processing is key for gene regulation. Using live-cell photobleaching techniques, we investigated the factor TCERG1, which coordinates transcriptional elongation with splicing. We demonstrate that TCERG1 is highly mobile in the nucleoplasm and that this mobility is slightly decreased when it is associated with speckles. Dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB) but not α-amanitin treatment reduced the mobility of TCERG1, which suggests interaction with paused transcription elongation complexes. We found that TCERG1 mobility is rapid at the transcription site (TS) of a reporter that splices post-transcriptionally and that TCERG1 is recruited to the active TS independent of the CTD of RNAPII, thus excluding phosphorylated CTD as a requirement for recruiting this factor to the TS. Importantly, the mobility of TCERG1 is reduced when the reporter splices cotranscriptionally, which suggests that TCERG1 forms new macromolecular complexes when splicing occurs cotranscriptionally. In this condition, spliceostatin A has no effect, indicating that TCERG1 rapidly binds and dissociates from stalled spliceosomal complexes and that the mobility properties of TCERG1 do not depend on events occurring after the initial spliceosome formation. Taken together, these data suggest that TCERG1 binds independently to elongation and splicing complexes, thus performing their coupling by transient interactions rather than by stable association with one or the other complexes. This finding has conceptual implications for understanding the coupling between transcription and RNA processing.

Prp40 and early events in splice site definition.

The alternative splicing (AS) of precursor messenger RNA (pre-mRNA) is a tightly regulated process through which introns are removed to leave the resulting exons in the mRNA appropriately aligned and ligated. The AS of pre-mRNA is a key mechanism for increasing the complexity of proteins encoded in the genome. In humans, more than 90% of genes undergo AS, underscoring the importance of this process in RNA biogenesis. As such, AS misregulation underlies multiple human diseases. The splicing reaction is catalyzed by the spliceosome, a highly dynamic complex that assembles at or near the intron/exon boundaries and undergoes sequential conformational and compositional changes during splicing. The initial recognition of splice sites defines the exons that are going to be removed, which is a critical step in the highly regulated splicing process. Although the available lines of evidence are increasing, the molecular mechanisms governing AS, including the initial interactions occurring at intron/exon boundaries, and the factors that modulate these critical connections by functioning as a scaffold for active-site RNAs or proteins, remain poorly understood. In this review, we summarize the major hallmarks of the initial steps in the splicing process and the role of auxiliary factors that contribute to the assembly of the spliceosomal complex. We also discuss the role of the essential yeast Prp40 protein and its mammalian homologs in the specificity of this pre-mRNA processing event. In addition, we provide the first exhaustive phylogenetic analysis of the molecular evolution of Prp40 family members. WIREs RNA 2016, 7:17-32. doi: 10.1002/wrna.1312 For further resources related to this article, please visit the WIREs website.

Functional Consequences for Apoptosis by Transcription Elongation Regulator 1 (TCERG1)-Mediated Bcl-x and Fas/CD95 Alternative Splicing.

Here, we present evidence for a specific role of the splicing-related factor TCERG1 in regulating apoptosis in live cells by modulating the alternative splicing of the apoptotic genes Bcl-x and Fas. We show that TCERG1 modulates Bcl-x alternative splicing during apoptosis and its activity in Bcl-x alternative splicing correlates with the induction of apoptosis, as determined by assessing dead cells, sub-G1-phase cells, annexin-V binding, cell viability, and cleavage of caspase-3 and PARP-1. Furthermore, the effect of TCERG1 on apoptosis involved changes in mitochondrial membrane permeabilization. We also found that depletion of TCERG1 reduces the expression of the activated form of the pro-apoptotic mitochondrial membrane protein Bak, which remains inactive by heterodimerizing with Bcl-xL, preventing the initial step of cytochrome c release in Bak-mediated mitochondrial apoptosis. In addition, we provide evidence that TCERG1 also participates in the death receptor-mediated apoptosis pathway. Interestingly, TCERG1 also modulates Fas/CD95 alternative splicing. We propose that TCERG1 sensitizes a cell to apoptotic agents, thus promoting apoptosis by regulating the alternative splicing of both the Bcl-x and Fas/CD95 genes. Our findings may provide a new link between the control of alternative splicing and the molecular events leading to apoptosis.

Recent insights into the transcriptional control of the Tcra/Tcrd locus by distant enhancers during the development of T-lymphocytes.

Tcra/Tcrd includes 2 genes with distinct developmental programs controlled by 2 distant enhancers, Eα and Eδ. These enhancers work as a developmental switch during thymocyte development and they are essential for generation of αß and γδ T-lymphocytes. Tcra and Tcrd transit from an unrearranged configuration to a rearranged configuration during T-cell development. Eα and Eδ are responsible for transcription of their respective unrearranged genes in thymocytes but are dispensable for such functions in the context of the rearranged genes in mature T-cells. Interestingly, Eα activates transcription of the rearranged Tcrd in γδ T-lymphocytes but it is inactive in αß T-lymphocytes.

T-cell receptor α enhancer is inactivated in αß T lymphocytes.

The Tcra enhancer (Eα) is essential for Tcra locus germ-line transcription and primary Vα-to-Jα recombination during thymocyte development. We found that Eα is inhibited late during thymocyte differentiation and in αß T lymphocytes, indicating that it is not required to drive transcription of rearranged Tcra genes. Eα inactivation resulted in the disruption of functional long-range enhancer-promoter interactions and was associated with loss of Eα-dependent histone modifications at promoter and enhancer regions, and reduced expression and recruitment of E2A to the Eα enhanceosome in T cells. Enhancer activity could not be recovered by T-cell activation, by forced expression of E2A or by the up-regulation of this and other transcription factors in the context of T helper differentiation. Our results argue that the major function of Eα is to coordinate the formation of a chromatin hub that drives Vα and Jα germ-line transcription and primary rearrangements in thymocytes and imply the existence of an Eα-independent mechanism to activate transcription of the rearranged Tcra locus in αß T cells.