Target Gene Expression In Cells Research Articles

Enhancing extracellular vesicle cargo loading and functional delivery by engineering protein-lipid interactions

Naturally generated lipid nanoparticles termed extracellular vesicles (EVs) hold significant promise as engineerable therapeutic delivery vehicles. However, active loading of protein cargo into EVs in a manner that is useful for delivery remains a challenge. Here, we demonstrate that by rationally designing proteins to traffic to the plasma membrane and associate with lipid rafts, we can enhance loading of protein cargo into EVs for a set of structurally diverse transmembrane and peripheral membrane proteins. We then demonstrate the capacity of select lipid tags to mediate increased EV loading and functional delivery of an engineered transcription factor to modulate gene expression in target cells. We envision that this technology could be leveraged to develop new EV-based therapeutics that deliver a wide array of macromolecular cargo.

Exploring the Role of Extracellular Vesicles in Skeletal Muscle Regeneration.

Skeletal muscle regeneration entails a multifaceted process marked by distinct phases, encompassing inflammation, regeneration, and remodeling. The coordination of these phases hinges upon precise intercellular communication orchestrated by diverse cell types and signaling molecules. Recent focus has turned towards extracellular vesicles (EVs), particularly small EVs, as pivotal mediators facilitating intercellular communication throughout muscle regeneration. Notably, injured muscle provokes the release of EVs originating from myofibers and various cell types, including mesenchymal stem cells, satellite cells, and immune cells such as M2 macrophages, which exhibit anti-inflammatory and promyogenic properties. EVs harbor a specific cargo comprising functional proteins, lipids, and nucleic acids, including microRNAs (miRNAs), which intricately regulate gene expression in target cells and activate downstream pathways crucial for skeletal muscle homeostasis and repair. Furthermore, EVs foster angiogenesis, muscle reinnervation, and extracellular matrix remodeling, thereby modulating the tissue microenvironment and promoting effective tissue regeneration. This review consolidates the current understanding on EVs released by cells and damaged tissues throughout various phases of muscle regeneration with a focus on EV cargo, providing new insights on potential therapeutic interventions to mitigate muscle-related pathologies.

Extracellular Vesicles and Immunity: At the Crossroads of Cell Communication.

Extracellular vesicles (EVs), comprising exosomes and microvesicles, are small membranous structures secreted by nearly all cell types. They have emerged as crucial mediators in intercellular communication, playing pivotal roles in diverse physiological and pathological processes, notably within the realm of immunity. These roles go beyond mere cellular interactions, as extracellular vesicles stand as versatile and dynamic components of immune regulation, impacting both innate and adaptive immunity. Their multifaceted involvement includes immune cell activation, antigen presentation, and immunomodulation, emphasising their significance in maintaining immune homeostasis and contributing to the pathogenesis of immune-related disorders. Extracellular vesicles participate in immunomodulation by delivering a wide array of bioactive molecules, including proteins, lipids, and nucleic acids, thereby influencing gene expression in target cells. This manuscript presents a comprehensive review that encompasses in vitro and in vivo studies aimed at elucidating the mechanisms through which EVs modulate human immunity. Understanding the intricate interplay between extracellular vesicles and immunity is imperative for unveiling novel therapeutic targets and diagnostic tools applicable to various immunological disorders, including autoimmune diseases, infectious diseases, and cancer. Furthermore, recognising the potential of EVs as versatile drug delivery vehicles holds significant promise for the future of immunotherapies.

P-054 Significant differences of micro RNA expression pattern in extracellular vesicles between fertile and infertile individuals: new markers of male reproductive status?

Abstract Study question Is the miRNAs cargo of the extracellular vesicles (EVs) present in seminal plasma different between healthy semen donors and infertile patients? Summary answer There are significant differences in the miRNAs expression patterns in the load of seminal EVs between infertile patients and healthy semen donors What is known already Extracellular vesicles (EVs) present in seminal plasma contain a wide range of different proteins, lipids and nucleic acids, such as microRNA. These microRNA can execute their action by regulating gene expression of target cells triggering an important role in different reproductive processes. It is well stablished in different pathologies that the EVs cargo is representative for the functional status of the producer cell. In this sense, the alteration in microRNAs expression patterns in seminal plasma has been associated with spermatogenic impairments, subfertility and azoospermia, indicating that microRNAs in seminal EVs could work as biomarkers of the male fertility status Study design, size, duration This prospective study, performed between March 2021 to December 2022, included 30 semen samples from infertile males seeking infertility treatment (study group), and 7 semen samples from healthy semen donors (control group) from our donor semen bank. All the semen samples from patients showed abnormal seminal parameters (oligo- and/or asthenozoospermia). Men under drug treatment and/or with infectious or chronic diseases were excluded from the study Participants/materials, setting, methods After conventional semen analysis, EVs were isolated from seminal plasma by ultracentrifugation. To confirm EVs isolation, an aliquot of each sample was used for nanoparticle tracking analysis (NTA). Then, microRNAs from EVs were extracted and miRNAseq was performed. Raw data were standardized using counts per millions and ANOVA was performed to assess differences in the miRNAs expression between groups.Target genes were annotated using miRTarBase and functional enrichment analysis was performed by MIENTURNET and GeneCards Main results and the role of chance The presence of EVs after ultracentrifugation of seminal plasma was confirmed by NTA, showing a range size within 40-140 nm. miRNAseq generated reads for 914 annotated miRNAs and significant differences in the expression of 17 of them were found between patient and control samples. These miRNAs target a total of 1310 genes and functional enrichment analysis indicated that they participate in several processes related to cell differentiation, cell proliferation, immunomodulation, cellular adhesion and apoptosis. Specifically, patient group showed significant higher expression of some members of the hsa-let-7 family (p < 0.001 for let-7i-5p, let-7f-5p, let-7c-5p and let-7a-5p; p < 0.05 for let-7b-5p), which are implicated in adaptative immune system activation and differentiation by gene modulation. Results also found significant (p < 0.01) higher expression in patient group of hsa-miR-92a-3p, hsa-miR-200c-3p and hsa-miR-888-5p, which target genes are related to apoptotic processes. Finally, patient group showed significant (p < 0.001) lower expression of hsa-miR-320b and miR-320a-3p, which interact with transcription factor sequences (DLX5, MYC) implicated in cell proliferation and differentiation; and also lower expression in hsa-miR-423-5p (p < 0.01) and miR-423-3p (p < 0.05) that interact with the sequence of transcription factors as p21, a regulator of cell cycle progression at G1, and with RNA-binding proteins involved in ribosome assembly. Limitations, reasons for caution This study included males with dissimilar seminal disorders (astheno, oligo, and teratozoospermia). Therefore, further studies are needed lowering the heterogeneity of the study group. At present, no data about reproductive outcome is available Wider implications of the findings This work suggests that the significant differences found in the expression pattern in microRNAs in seminal EVs between infertile and fertile men may be related with gene modulation in the reproductive processes and could be used as novel biomarkers of male fertility status. Trial registration number 1904-MAD-045-AP

Extracellular vesicles: pathogenic messengers and potential therapy for neonatal lung diseases.

Extracellular vesicles (EVs) are a heterogeneous group of nano-sized membranous structures increasingly recognized as mediators of intercellular and inter-organ communication. EVs contain a cargo of proteins, lipids and nucleic acids, and their cargo composition is highly dependent on the biological function of the parental cells. Their cargo is protected from the extracellular environment by the phospholipid membrane, thus allowing for safe transport and delivery of their intact cargo to nearby or distant target cells, resulting in modification of the target cell's gene expression, signaling pathways and overall function. The highly selective, sophisticated network through which EVs facilitate cell signaling and modulate cellular processes make studying EVs a major focus of interest in understanding various biological functions and mechanisms of disease. Tracheal aspirate EV-miRNA profiling has been suggested as a potential biomarker for respiratory outcome in preterm infants and there is strong preclinical evidence showing that EVs released from stem cells protect the developing lung from the deleterious effects of hyperoxia and infection. This article will review the role of EVs as pathogenic messengers, biomarkers, and potential therapies for neonatal lung diseases.

Subviral Dense Bodies of Human Cytomegalovirus Induce an Antiviral Type I Interferon Response.

(1) Background: Cells infected with the human cytomegalovirus (HCMV) produce subviral particles, termed dense bodies (DBs), both in-vitro and in-vivo. They are released from cells, comparable to infectious virions, and are enclosed by a membrane that resembles the viral envelope and mediates the entry into cells. To date, little is known about how the DB uptake influences the gene expression in target cells. The purpose of this study was to investigate the impact of DBs on cells, in the absence of a viral infection. (2) Methods: Mass spectrometry, immunoblot analyses, siRNA knockdown, and a CRISPR-CAS9 knockout, were used to investigate the changes in cellular gene expression following a DB exposure; (3) Results: A number of interferon-regulated genes (IRGs) were upregulated after the fibroblasts and endothelial cells were exposed to DBs. This upregulation was dependent on the DB entry and mediated by the type I interferon signaling through the JAK-STAT pathway. The induction of IRGs was mediated by the sensing of the DB-introduced DNA by the pattern recognition receptor cGAS. (4) Conclusions: The induction of a strong type I IFN response by DBs is a unique feature of the HCMV infection. The release of DBs may serve as a danger signal and concomitantly contribute to the induction of a strong, antiviral immune response.

Why the complications of COVID-19 patients differ in elderly and young cancer patients

Zhang et al. reported the impact of different risk factors and comorbidities in COVID-19 lethality. The authors observed that the odds of dying by COVID-19 in cancer patients decrease with age and cancer becomes a non-significant factor above 80 years. We speculate on the possible causes for the different COVID-19 severity between elderly and young patients. Several factors that can have a different impact on young and elderly have to be taken into account such as inflammation, microbiota and anti-cancer therapies. Inflammaging is a complex process that characterizes elderly people and it is believed to contribute to the severity of COVID-19 associated with old age. Cancer and related therapies may alter the process of inflammaging both quantitatively and qualitatively and could impact on COVID-19 severity. Moreover, therapies used in elderly cancer patients are usually different from that used for young people where the presence of comorbidities and the mechanisms of action of the different drugs both on the susceptibility genes and on other factors have to be considered. Sex hormones and anti-estrogen therapies affect significantly gene expression in target cells thereby modulating the susceptibility of the tissues to SARS-CoV-2 infection and as a consequence the extent of the symptoms. The concentration of sex hormones varies with aging and among sexes. Interestingly, recent evidences, further corroborate the hypothesis that also sex hormones or anti-estrogen therapies impact the susceptibility to COVID-19 and its severity.

IMP1/IGF2BP1 alters extracellular vesicle cargo in colorectal cancer

BackgroundColorectal cancer (CRC) is the third leading cause of cancer‐related death worldwide. There is an urgent need for new therapeutic targets and methods of early detection to improve patient outcomes. Extracellular Vesicles (EVs) and EV cargo are promising new biomarkers and therapeutic targets. EVs are secreted nanoparticles that carry DNA, RNA, and proteins. EVs can target specific cell types and once taken up by a destination cell, EV cargo can shape cell gene expression and physiology. EVs can shape the tumor microenvironment (TME) (i.e., stromal, immune, and endothelial cells), which plays critical roles in patient survival and disease outcomes. Our current understanding of how EV cargo is determined and how it influences the CRC cell TME is limited. One promising but understudied molecular aspect of EVs is RNA binding proteins (RNAbps). RNAbps are post‐transcriptional regulators of gene expression and could provide a link between host cell gene expression and EV cargo. Insulin‐like growth factor 2 RNA binding protein 1 (IMP1, IGF2BP1) is an RNAbp with a critical role in CRC development and progression. IMP1 is expressed highly in CRC tumors, expression correlates with worse prognosis, and IMP1 binds/potently regulates tumor‐associated transcripts. How EV cargo is influenced by IMP1 and effects on EV targeting to destination cells and on target cell gene expression and function is not known. Understanding how IMP1 shapes EV cargo and which TME cells are targeted, will identify new mechanisms of cancer development/progression through IMP1‐EV‐mediated alteration of the TME and novel EV cargo as candidates for early detection biomarkers.HypothesisWe hypothesize that IMP1 facilitates the packaging of tumor‐promoting EV cargo that interact and communicate with the TME to enhance tumor progression.MethodsEVs were isolated from IMP1 null and IMP1‐overexpressing (IMP1OE) CRC cell lines, HT‐29, SW480, as well as wildtype Caco2 cells (express high levels of IMP1) by size‐exclusion chromatography. EVs were assessed by nanoparticle tracking analysis, western blot, electron microscopy, and qPCR.ResultsWe found that IMP1 enters EVs from Caco2 and SW480 IMP1OE cell lines, but not HT‐29 IMP1OE or corresponding null controls. IMP1 did not alter EV secretion in CRC cell lines. We found that IMP1 target mRNAs, such as KRAS, cMYC are present within EVs, but not the IMP1 target PTGS2.ConclusionsOur novel findings suggest that IMP1 enters the EV to directly influence EV cargo in specific CRC cell lines. Our findings have implications for the development of novel early detection biomarkers and therapeutic approaches in CRC.

Exosomes in the Pathogenesis, Progression, and Treatment of Osteoarthritis.

Osteoarthritis (OA) is a prevalent and debilitating age-related joint disease characterized by articular cartilage degeneration, synovial membrane inflammation, osteophyte formation, as well as subchondral bone sclerosis. OA drugs at present are mainly palliative and do not halt or reverse disease progression. Currently, no disease-modifying OA drugs (DMOADs) are available and total joint arthroplasty remains a last resort. Therefore, there is an urgent need for the development of efficacious treatments for OA management. Among all novel pharmaco-therapeutical options, exosome-based therapeutic strategies are highly promising. Exosome cargoes, which include proteins, lipids, cytokines, and various RNA subtypes, are potentially capable of regulating intercellular communications and gene expression in target cells and tissues involved in OA development. With extensive research in recent years, exosomes in OA studies are no longer limited to classic, mesenchymal stem cell (MSC)-derived vesicles. New origins, structures, and functions of exosomes are constantly being discovered and investigated. This review systematically summarizes the non-classic origins, biosynthesis, and extraction of exosomes, describes modification and delivery techniques, explores their role in OA pathogenesis and progression, and discusses their therapeutic potential and hurdles to overcome in OA treatment.

Характеристика полиморфизмов гена рецептора витамина D

The article presents the characteristics of the main vitamin D receptor (VDR) gene polymorphisms: rs2228570 (FokI), rs731236 (TaqI), rs1544410 (BsmI) and rs7975232 (ApaI). The role of the vitamin D hormonally active form (1,25(OH)2D3, calcitriol) as a transcription factor regulating gene expression in target cells by binding to the vitamin D receptor protein is described. The immunomodulatory and mediating effect of VDRs on the biological functions of the human body has been noted. A description of the vitamin D receptor gene and its polymorphic character have been provided. The analysis of the four most significant single nucleotide polymorphisms (SNPs) of the VDR gene was carried out. A detailed description of each polymorphism, its genomic position, the nature of interaction with other polymorphisms of the vitamin D receptor gene, as well as its effect on the structure and activity of the VDR protein were given. The analysis of the indicated single-nucleotide polymorphisms allelic composition was conducted according to the literature and specialized SNP databases. The frequency of each polymorphism individual alleles occurrence, as well as their influence on the predisposition and course of various diseases, were studied. The need for further studies of VDR gene polymorphisms, their allelic composition and prevalence was designated. It is also necessary to study the possibilities of their potential use as genetic markers for such relevant but little-studied pathologies as COVID-19.

A portable system for isothermal amplification and detection of exosomal microRNAs

Exosomal microRNAs (miRNAs) play a key role in cell-cell communication to regulate gene expression in target cells and have great potential as biomarkers for disease diagnosis. This paper reports an on-chip exosomal miRNA amplification and detection system for rapid analysis of exosomal miRNAs. The compact system consists of two connected flow cells for processing exosomes and detecting miRNAs, respectively. The miRNAs extracted from exosomes were quantitatively measured using the on-chip exponential amplification reaction (EXPAR) assay. The sensor chip was designed to store multiple oligonucleotide templates for the EXPAR, mix sample and reagent, and simultaneously analyze multiple exosomal miRNAs of interest. To facilitate the miRNA analysis, a portable detection instrument was built on an IoT platform using a low-cost microcontroller to execute the EXPAR assay, collect fluorescent images, and analyze amplification curves. Here, we studied the miRNA profiles carried by exosomes derived from three different phenotypes of tissue macrophages. The affordable instrument, rapid assay, multiplexed analysis, as well as disposable sensor chip, would boost the development of point-of-care liquid biopsy tests using exosomal miRNAs.

Muscle-specific deletion of the vitamin D receptor in mice is associated with diaphragm muscle weakness.

Diseases or conditions where diaphragm muscle (DIAm) function is impaired, including chronic obstructive pulmonary disease, cachexia, asthma, and aging, are associated with an increased risk of pulmonary symptoms, longer duration of hospitalizations, and increasing requirements for mechanical ventilation. Vitamin D deficiency is associated with proximal muscle weakness that resolves following therapy with vitamin D3. Skeletal muscle expresses the vitamin D receptor (VDR), which responds to the active form of vitamin D, 1,25-dihydroxyvitamin D3 by altering gene expression in target cells. In knockout mice without skeletal muscle VDRs, there is marked atrophy of muscle fibers and a change in skeletal muscle biochemistry. We used a tamoxifen-inducible skeletal muscle Cre recombinase in Vdrfl/fl mice (Vdrfl/fl actin.iCre+) to assess the role of muscle-specific VDR signaling on DIAm-specific force, fatigability, and fiber type-dependent morphology. Vdrfl/fl actin.iCre+ mice treated with vehicle and Vdrfl/fl mice treated with tamoxifen served as controls. Seven days following the final treatment, mice were euthanized, the DIAm was removed, and isometric force and fatigue were assessed in DIAm strips using direct muscle stimulation. The proportion and cross-sectional areas of DIAm fiber types were evaluated by immunolabeling with myosin heavy chain antibodies differentiating type I, IIa and IIx, and/or IIb fibers. We show that in mice with skeletal muscle-specific VDR deletion, maximum specific force and residual force following fatigue are impaired, along with a selective atrophy of type IIx and/or IIb fibers. These results show that the VDR has a significant biological effect on DIAm function independent of systemic effects on mineral metabolism.NEW & NOTEWORTHY Vitamin D deficiency and vitamin D receptor (VDR) polymorphisms are associated with adverse pulmonary and diaphragm muscle (DIAm)-associated respiratory outcomes. We used a skeletal muscle-specific tamoxifen-inducible VDR knockout to investigate DIAm dysfunction following reduced VDR signaling. Marked DIAm weakness and atrophy of type IIx and/or IIb fibers are present in muscle-specific tamoxifen-induced VDR knockout mice compared with controls. These results show that the VDR has a significant biological effect on DIAm function independent of systemic effects on mineral metabolism.

MicroRNAs from Extracellular Vesicles Secreted by Bovine Embryos as Early Biomarkers of Developmental Competence.

During early development, embryos secrete extracellular vesicles (EVs) that participate in embryo–maternal communication. Among other molecules, EVs carry microRNAs (miRNAs) that interfere with gene expression in target cells; miRNAs participate in embryo–maternal communication. Embryo selection based on secreted miRNAs may have an impact on bovine breeding programs. This research aimed to evaluate the size, concentration, and miRNA content of EVs secreted by bovine embryos with different developmental potential, during the compaction period (days 3.5–5). Individual culture media from in vitro–produced embryos were collected at day 5, while embryos were further cultured and classified at day 7, as G1 (conditioned-culture media by embryos arrested in the 8–16-cells stage) and G2 (conditioned-culture media by embryos that reached blastocyst stages at day 7). Collected nanoparticles from embryo conditioned culture media were cataloged as EVs by their morphology and the presence of classical molecular markers. Size and concentration of EVs from G1 were higher than EVs secreted by G2. We identified 95 miRNAs; bta-miR-103, bta-miR-502a, bta-miR-100, and bta-miR-1 were upregulated in G1, whereas bta-miR-92a, bta-miR-140, bta-miR-2285a, and bta-miR-222 were downregulated. The most significant upregulated pathways were fatty acid biosynthesis and metabolism, lysine degradation, gap junction, and signaling pathways regulating pluripotency of stem cells. The characteristics of EVs secreted by bovine embryos during the compaction period vary according to embryo competence. Embryos that reach the blastocyst stage secrete fewer and smaller vesicles. Furthermore, the loading of specific miRNAs into the EVs depends on embryo developmental competence.

Characterization of promoters for adeno-associated virus mediated efficient Cas9 activation in adult Cas9 knock-in murine cochleae

CRISPR/Cas9 gene editing enables the treatment of hearing loss in congenitally deaf neonatal mice via both viral and non-viral delivery. While adeno-associated virus (AAV)-mediated gene delivery systems have been shown to be effective tools for gene replacement in the inner ear, application of the AAV-mediated CRISPR/Cas9 gene-editing approach for this purpose is yet to be documented. Based on our previous findings, we focused on the effects of several AAVs delivered via canalostomy injection in adult mice. Among the AAVs examined, AAV8 showed the greatest efficiency and specificity in transducing inner hair cells (IHC). The ability of Cre-expressing AAV8 to activate Cas9 in floxed-Cas9 knock-in (Cas9 KI) mice was further evaluated. We compared the effects of six different promoters (CMV, CAG, hSyn, CaMKIIa, GFAP, and ALB) of AAV8 delivered to the inner ear of adult Cas9 KI mice. Our findings showed that three AAV groups (CMV, CAG and hSyn promoters) infected the inner ear efficiently with different tropisms. Notably, AAVs with CMV, CAG, and hSyn promoters infected diverse cell types in mature murine cochleae, including IHCs. In particular, AAV8-hSyn showed high affinity to IHCs and spiral ganglion neurons (SGN). Neither the AAV8 virus itself (except AAV8-CAG) nor the surgical procedures used caused damage to HCs or impaired normal hearing. Our findings indicated that injection of AAV-Cre into mature inner ear efficiently induces Cas9 activation to achieve safe and efficient gene editing and different constituent promoters confer diverse infection patterns in cochlea, expanding the repertoire of gene-editing tools for regulating gene expression in target cells of the inner ear as part of the collective effort to rescue genetic hearing loss and develop effective gene therapy techniques.

Targeting the pregnane X receptor using microbial metabolite mimicry.

The human PXR (pregnane X receptor), a master regulator of drug metabolism, has essential roles in intestinal homeostasis and abrogating inflammation. Existing PXR ligands have substantial off‐target toxicity. Based on prior work that established microbial (indole) metabolites as PXR ligands, we proposed microbial metabolite mimicry as a novel strategy for drug discovery that allows exploiting previously unexplored parts of chemical space. Here, we report functionalized indole derivatives as first‐in‐class non‐cytotoxic PXR agonists as a proof of concept for microbial metabolite mimicry. The lead compound, FKK6 (Felix Kopp Kortagere 6), binds directly to PXR protein in solution, induces PXR‐specific target gene expression in cells, human organoids, and mice. FKK6 significantly represses pro‐inflammatory cytokine production cells and abrogates inflammation in mice expressing the human PXR gene. The development of FKK6 demonstrates for the first time that microbial metabolite mimicry is a viable strategy for drug discovery and opens the door to underexploited regions of chemical space.

Short non-coding RNA sequencing of glioblastoma extracellular vesicles.

Like all nucleated cells, glioblastoma (GBM) cells shed small membrane-encapsulated particles called extracellular vesicles (EVs). EVs can transfer oncogenic components and promote tumor growth by transferring short non-coding RNAs, altering target cell gene expression. Furthermore, GBM-derived EVs can be detected in blood and have potential to serve as liquid biopsies. EVs were harvested from culture supernatants from human GBM cell lines, purified via sequential centrifugation, and quantified by nanoparticle tracking. RNA was isolated and short non-coding RNA was sequenced. Data was analyzed via the OASIS-2.0 platform using HG38. MirTarBase and MirDB interrogated validated/predicted miRNA-gene interactions respectively. Many short non-coding RNA's were identified within GBM EV's. In keeping with earlier reports utilizing GBM EV micro-RNA (miRNA) arrays, these included abundant micro-RNA's including miR-21. However, RNA sequencing revealed a total of 712 non-coding RNA sequences most of which have not been associated with GBM EV's previously. These included many RNA species (piRNA, snoRNA, snRNA, rRNA and yRNAs) in addition to miRNA's. miR-21-5p, let-7b-5p, miR-3182, miR-4448, let-7i-5p constituted highest overall expression. Top genes targeted by non-coding RNA's were highly conserved and specific for cell cycle, PI3K/Akt signaling, p53 and Glioma curated KEGG pathways. Next generation short non-coding RNA sequencing on GBM EV's validates findings from earlier studies using miRNA arrays but also demonstrates expression of many additional non-coding RNA sequences and classes previously unassociated with GBM. This may yield important insights into pathophysiology, point to new therapeutic targets, and help develop new biomarkers for disease burden and treatment response.

New Potent DOT1L Inhibitors for in Vivo Evaluation in Mouse.

In MLL-rearranged cancer cells, disruptor of telomeric silencing 1-like protein (DOT1L) is aberrantly recruited to ectopic loci leading to local hypermethylation of H3K79 and consequently misexpression of leukemogenic genes. A structure-guided optimization of a HTS hit led to the discovery of DOT1L inhibitors with subnanomolar potency, allowing testing of the therapeutic principle of DOT1L inhibition in a preclinical mouse tumor xenograft model. Compounds displaying good exposure in mouse and nanomolar inhibition of target gene expression in cells were obtained and tested in vivo.

79 MicroRNAs of extracellular vesicles secreted by embryos as an early biomarker of competence

Extracellular vesicles (EVs), including exosomes and microvesicles, are secreted by different cell types and participate in cellular communication by carrying molecules as microRNAs (miRNAs) that can interfere with gene expression of target cells. Extracellular vesicles have become relevant as a mechanism of embryo-maternal communication. The aim of this study was to evaluate miRNA content in EVs secreted after embryonic genome activation, by bovine embryos with different developmental potential. Bovine embryos were produced invitro and cultured in group until Day 3.5 in synthetic oviductal fluid (SOF) medium. Only 8-16-cell embryos were cultured individually in EVs-depleted SOF until Day 5. The SOF was EV depleted by ultrafiltration. Culture media (CM) were collected at Day 5 and embryos continued in culture until Day 7 with fresh SOF. Collected media were conserved individually and identified with the corresponding embryo. Then, CM were classified according to capacity of its embryo to reach blastocyst stage at Day 7: G1-CM (blocked embryos in 8-16 cell) and G2-CM (embryos that reach blastocyst stage). The EV isolation was carried out using the protocol described by Mellisho et al. (2017). Recovered EVs were evaluated by nanoparticle tracking analysis (NTA), Transmission electron microscopy and the presence of surface markers (CD9, CD63, CD81, and CD40L). After NTA, individual CM were pooled to organise 3 replicates of 10CM each, for G1 and G2. The whole miRNA isolation, library preparation, and sequencing was performed by Norgen Biotek facilities (Canada). The quality of libraries was analysed using the FastQC program platform followed by Trimmomatic to remove remnant adapters. For the miRNA library it accepted reads with value above 30 Phreads and 22 to 30bp length. The reads were mapped against the reference genome ARS-UCD1.2 using Bowtie2 software and miRDeep2 mapper, and the gene counts were calculated using HTSeq. Differential expression analysis was performed in EdgeR package. To expand this information, principal component analysis, Heatmap, and Volcano plot were plotted and pathway enrichment analysis was conducted. The NTA, transmission electron microscopy, and flow cytometry confirmed the presence of exosomes and microvesicles in isolated EVs. According to NTA, the mean size of EVs was 102.1-176.2nm and concentration of 8.4×107-8.6×108 particlesmL−1 in G1 and G2, respectively. We identified 96 miRNAs significantly expressed across the samples. Only eight miRNAs in EVs were differentially expressed between groups (G2 vs. G1). The bta-miR-103, bta-miR-502a, bta-miR-100, and bta-miR-1 were up-regulated (Log2 fold-change>1), whereas bta-miR-92a, bta-miR-140, bta-miR-2285a, and bta-miR-222 were down-regulated (Log2 fold-change<1). The more significant (P-value<0.01) up-regulated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were fatty acid biosynthesis and metabolism, lysine degradation, gap junction, and signaling pathways regulating pluripotency of stem cells. The EVs secreted by embryos to culture environment carry miRNAs that can reflect the molecular state of their parental cell. This lets us suggest culture media derived-EVs and their miRNA cargo as early biomarkers to select more competent bovine embryos. This research was supported by FONDECYT, Chile (1170310).

Phase I/II Manufacture of Lentiviral Vectors Under GMP in an Academic Setting.

In clinical gene transfer applications, lentiviral vectors (LV) have rapidly become the primary means to achieve permanent and stable expression of a gene of interest or alteration of gene expression in target cells. This status can be attributed primarily to the ability of the LV to (1) transduce dividing as well as quiescent cells, (2) restrict or expand tropism through envelope pseudo-typing, and (3) regulate gene expression within different cell lineages through internal promoter selection. Recent progress in viral vector design such as the elimination of unnecessary viral elements, split packaging, and self-inactivating vectors has established a significant safety profile for these vectors. The level of GMP compliance required for the manufacture of LV is dependent upon their intended use, stage of drug product development, and country where the vector will be used as the different regulatory authorities who oversee the clinical usage of such products may have different requirements. As such, successful GMP manufacture of LV requires a combination of diverse factors including: regulatory expertise, compliant facilities, validated and calibrated equipments, starting materials of the highest quality, trained production personnel, scientifically robust production processes, and a quality by design approach. More importantly, oversight throughout manufacturing by an independent Quality Assurance Unit who has the authority to reject or approve the materials is required. We describe here the GMP manufacture of LV at our facility using a four plasmid system where 293T cells from an approved Master Cell Bank (MCB) are transiently transfected using polyethylenimine (PEI). Following transfection, the media is changed and Benzonase added to digest residual plasmid DNA. Two harvests of crude supernatant are collected and then clarified by filtration. The clarified supernatant is purified and concentrated by anion exchange chromatography and tangential flow filtration. The final product is then diafiltered directly into the sponsor defined final formulation buffer and aseptically filled.

Exosomes in intercellular communication and implications for osteoarthritis.

Osteoarthritis (OA) is the most prevalent of the musculoskeletal conditions and represents a significant public health burden. While degeneration of articular cartilage is a key feature, it is now increasingly recognized as a complex condition affecting the whole joint, with synovial inflammation present in a significant proportion of patients. As a secretory tissue, the OA synovium is a rich source of both soluble inflammatory mediators and extracellular vesicles, including exosomes, which have been implicated in cell-cell communication. Exosome cargo has been found to include proteins, lipids and various RNA subtypes such as mRNA and miRNA, potentially capable of regulating gene expression in target cells and tissues. Profiling of exosome cargo and understanding effects on cartilage could elucidate novel regulatory mechanisms within the joint, providing insight for targeted treatment. The aim of this article is to review current literature on exosome biology, highlighting the relevance and application for OA pathogenesis.