Abstract Long-range functional enhancer–promoter (E-P) interactions typically occur within topologically associating domains (TADs), which facilitate contacts while restricting inter-domain communication. Although approximately one-third of E-P interactions cross TAD boundaries, they are considered non-functional and are only rarely reported during embryonic development. Here, we investigated the activity of the strong Tcra enhancer (Eα) positioned at the boundary between a centromeric TAD containing the T-lineage-specific Tcra-Tcrd locus and a telomeric TAD encompassing broadly expressed Dad1 -to- Cdh24 genes. To directly assess Eα activity across TADs, we generated mice lacking Eα while preserving its associated CTCF-binding elements. As expected, Eα was required for T-cell specific Tcra-Tcrd transcription and normal T-cell development. In contrast, Eα did not activate Dad1 or Haus4 transcription in the telomeric TAD, indicating effective insulation by boundary elements. Unexpectedly, Eα selectively activated developmentally regulated Cdh24 expression, the most distal gene in the telomeric TAD, in thymocytes. These findings reveal a naturally occurring, functional cross-TAD E-P interaction during adult T-cell development, demonstrating that enhancers positioned at TAD boundaries can bypass topological insulation to selectively regulate distal gene expression.

Background and purpose: From the early use of liposomes in the 1960s to modern nucleic acid delivery platforms, nanotechnology has shown its value in drug delivery and diagnostic applications. A wide range of nanostructures has been investigated for drug delivery, including inorganic, polymeric and lipid-based nanomaterials. While some of these nanomaterials have reached more advanced stages of clinical development, some even reaching commercialization, new alternatives are continuously being explored. Approach: This review describes the different categories of nanomaterials being used as nanocarriers and provides concrete representative examples of commercially approved products. Key results: Current trends of nanotechnology developments in the pharmaceutical market largely focus on the treatment of cancer, infectious diseases, central nervous system disorders and cardiovascular diseases. At the same time, lipid nanoparticle platforms are gaining relevance in current development pipelines. However, some of the main challenges limiting clinical translation include manufacturability, scale-up, long-term stability, regulatory uncertainty, intellectual property issues and the lack of representative preclinical study models. Conclusion: Nanotechnology has become a fundamental breakthrough in medicine, especially for advanced therapies such as gene silencing or gene editing alternatives. All things considered, no universal nanocarrier fits all biomedical applications; the evolution of the field depends on several platforms demonstrating their individual potential.

BDNF signaling through its receptor TRKB plays a critical role in brain development, neuroplasticity, and homeostasis. Alternative splicing of the TRKB gene, NTRK2, generates either the full-length receptor (TRKB-FL) or a truncated isoform (TRKB-T1) that inhibits BDNF signaling and has been implicated in neurodegenerative diseases, psychiatric disorders, and cognitive impairments. Here, we show that PRPF40B, a splicing factor associated with neuronal dysfunction, promotes the production of the TRKB-FL isoform during neuronal differentiation. Silencing PRPF40B increases TRKB-T1 expression and impairs the expression of genes important for neuronal differentiation and synaptic plasticity, both in vitro and in vivo, during early embryogenesis. Our data thus identify PRPF40B as a key regulator of the balance between TRKB receptor isoforms, crucial for fine-tuning neuronal responses and for preventing neuroplasticity or survival impairments, providing also a mechanism for the role of PRPF40B in the pathogenesis of various human neurodegenerative diseases and psychiatric disorders.

The Blood-Brain Barrier (BBB) significantly impedes drug delivery to the central nervous system. Nanotechnology, especially surface-functionalized lipid nanoparticles, offers innovative approaches to overcome this barrier. However, choosing an effective functionalization strategy is challenging due to the lack of detailed comparative analysis in current literature. Our systematic review examined various functionalization strategies and their impact on BBB permeability from 2041 identified articles, of which 80 were included for data extraction. Peptides were the most common modification (18) followed by mixed strategies (12) proteins (9), antibodies (7), and other strategies (8). Interestingly, 26 studies showed BBB penetration with unmodified or modified nanoparticles using commonly applied strategies such as PEGylation or surfactant addition. Statistical analysis across 42 studies showed correlation between higher in vivo permeation improvements and nanoparticle type, size, and functionalization category. The highest ratios were found for nanostructured lipid carriers or biomimetic systems, in studies with particle sizes under 150 nm, and in those applying mixed functionalization strategies. The interstudy heterogeneity we observed highlights the importance of adopting standardized evaluation protocols to enhance comparability. Our systematic review aims to provide a comparative insight and identify future research directions in the development of more effective lipid nanoparticle systems for drug delivery to the brain to help improve the treatment of neurological and psychiatric disorders and brain tumours.

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.

Manufacturing lipid nanoparticles through microfluidic mixing can be approached from a Quality by Design perspective. Research involving critical process parameters seems to focus on the total flow and flow rate ratio, thus other process variables, such as dialysis, are underestimated. This study used a Design of Experiments to identify the influence of critical process parameters on particle size, polydispersity index, and zeta potential. A response surface Design of Experiments modeled the influence of: total flow (400 to 4000 mu L min-1); flow rate ratio (3 to 9) and dialysis (yes/no). Results suggest that dialysis is a crucial parameter that strongly influences particle size and zeta potential and moderately affects polydispersity index. The flow rate ratio's relevance decreases when dialysis is performed. As the purification method can change the influence of other process parameters, it should be an integrated part of the microfluidic manufacturing of lipid nanoparticles instead of an extra step.

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 αβ T lymphocytes. Despite its strongly reduced activity, Eα is still required for high TCRα transcription and expression of TCRαβ 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.

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.

The authors of this volume on Genome and Epigenetics dedicate this publication to the memory of Jose Luis Gomez Skarmeta (1966–2020), CSIC Research Professor at the Andalusian Centre for Developmental Biology / Centro Andaluz de Biologia del Desarrollo (CABD).

Prefoldin is a heterohexameric complex conserved from archaea to humans that plays a cochaperone role during the co-translational folding of actin and tubulin monomers. Additional functions of prefoldin have been described, including a positive contribution to transcription elongation and chromatin dynamics in yeast. Here we show that prefoldin perturbations provoked transcriptional alterations across the human genome. Severe pre-mRNA splicing defects were also detected, particularly after serum stimulation. We found impairment of cotranscriptional splicing during transcription elongation, which explains why the induction of long genes with a high number of introns was affected the most. We detected genome-wide prefoldin binding to transcribed genes and found that it correlated with the negative impact of prefoldin depletion on gene expression. Lack of prefoldin caused global decrease in Ser2 and Ser5 phosphorylation of the RNA polymerase II carboxy-terminal domain. It also reduced the recruitment of the CTD kinase CDK9 to transcribed genes, and the association of splicing factors PRP19 and U2AF65 to chromatin, which is known to depend on CTD phosphorylation. Altogether the reported results indicate that human prefoldin is able to act locally on the genome to modulate gene expression by influencing phosphorylation of elongating RNA polymerase II, and thereby regulating co-transcriptional splicing.

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.

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 αβ and γδ T lymphocytes. The TCRα, TCRβ, 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, TCRαβ, or TCRγδ which drive thymocyte maturation into αβ 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.

TCERG1 es una proteina implicada en la transcripcion y el splicing de los mRNAs y en el acoplamiento funcional de ambos procesos. En esta tesis doctoral hemos descubierto una nueva funcion para esta proteina: TCERG1 regula la homeostasis de los cuerpos de Cajal (CBs) y la biogenesis de las ribonucleoproteinas nucleares pequenas (small nuclear ribonucleoprotein, snRNPs), particulas imprescindibles para el proceso de splicing del pre-mRNA. Este estudio se inicio con la identificacion de la region de TCERG1 responsable de la union con NOLC1, una fosfoproteina nucleolar componente de los CBs. Hemos identificado que los dominios FF4 y FF5 de TCERG1 son los responsables de la interaccion entre TCERG1 y NOLC1 y que la inhibicion de TCERG1 afecta a la localizacion de NOLC1 en los CBs nucleares pero no a su localizacion en el nucleolo. En relacion a los CBs, mostramos que la inhibicion de TCERG1 afecta la integridad de estos organulos ya que su proteina marcadora, la coilina, con la que interacciona TCEG1, se dispersa por el nucleoplasma en numerosos pequenos granulos. Este efecto es especifico de TCERG1 ya que los CBs pueden formarse de nuevo tras la reexpresion controlada de TCERG1. Ademas, la inhibicion de TCERG1 no parece afectar de forma prominente a otros organulos nucleares como los speckles, gems o HLB. Paradojicamente, el efecto de disgregacion de los CBs puede ser observado tambien con la sobreexpresion saturante de TCERG1. En esta tesis hemos identificado tambien un papel importante de TCERG1 en la biogenesis de los snRNPs. Hemos observado que la inhibicion de TCERG1 afecta la union de las proteinas Sm a los RNAs pequenos nucleares ricos en uridinas (snRNAs), union necesaria para formar el complejo snRNP maduro. La formacion de este complejo tras la inhibicion de TCERG1 se ve tambien disminuida al monitorizar la expresion de una proteina de fusion GFP-SmB. El papel de TCERG1 en la formacion del complejo Sm-snRNP no parece implicar un efecto sobre la transcripcion de los snRNAs dado que, en las condiciones experimentales usadas, los niveles de los snRNAs endogenos no estan disminuidos. La interaccion de TCERG1 con factores implicados en la biogenesis de los snRNPs, como CRM1, Int11 o SMN, refuerzan un posible papel de TCERG1 en este proceso. Nuestros datos indican que TCERG1 juega un papel importante en la homeostasis de los CBs y en el ensamblaje de los complejos Sm-snRNAs durante la biogenesis de los snRNPs.

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.

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.

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.

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.