1,721,141 research outputs found
Interplay of TLR4 and SARS-CoV-2: Possible Involvement of microRNAs [Letter]
No full textNo abstract availabl
Combined approaches for increasing fetal hemoglobin (HbF) and de novo production of adult hemoglobin (HbA) in erythroid cells from β-thalassemia patients: treatment with HbF inducers and CRISPR-Cas9 based genome editing
No full textGenome editing (GE) is one of the most efficient and useful molecular approaches to correct the effects of gene mutations in hereditary monogenetic diseases, including beta-thalassemia. CRISPR-Cas9 gene editing has been proposed for effective correction of the beta-thalassemia mutation, obtaining high-level "de novo" production of adult hemoglobin (HbA). In addition to the correction of the primary gene mutations causing beta-thalassemia, several reports demonstrate that gene editing can be employed to increase fetal hemoglobin (HbF), obtaining important clinical benefits in treated beta-thalassemia patients. This important objective can be achieved through CRISPR-Cas9 disruption of genes encoding transcriptional repressors of.-globin gene expression (such as BCL11A, SOX6, KLF-1) or their binding sites in the HBG promoter, mimicking non-deletional and deletional HPFH mutations. These two approaches (beta-globin gene correction and genome editing of the genes encoding repressors of.-globin gene transcription) can be, at least in theory, combined. However, since multiplex CRISPR-Cas9 gene editing is associated with documented evidence concerning possible genotoxicity, this review is focused on the possibility to combine pharmacologically-mediated HbF induction protocols with the "de novo" production of HbA using CRISPR-Cas9 gene editing
Recent trends for novel options in experimental biological therapy of β-thalassemia
No full textINTRODUCTION:
β-thalassemias are caused by nearly 300 mutations of the β-globin gene, leading to low or absent production of adult hemoglobin. Achievements have been recently obtained on innovative therapeutic strategies for β-thalassemias, based on studies focusing on the transcriptional regulation of the γ-globin genes, epigenetic mechanisms governing erythroid differentiation, gene therapy and genetic correction of the mutations.
AREAS COVERED:
The objective of this review is to describe recently published approaches (the review covers the years 2011 - 2014) useful for the development of novel therapeutic strategies for the treatment of β-thalassemia.
EXPERT OPINION:
Modification of β-globin gene expression in β-thalassemia cells was achieved by gene therapy (eventually in combination with induction of fetal hemoglobin [HbF]) and correction of the mutated β-globin gene. Based on recent areas of progress in understanding the control of γ-globin gene expression, novel strategies for inducing HbF have been proposed. Furthermore, the identification of microRNAs involved in erythroid differentiation and HbF production opens novel options for developing therapeutic approaches for β-thalassemia and sickle-cell anemia
Ground state naïve pluripotent stem cells and CRISPR/Cas9 gene correction for β-thalassemia
No full textThe β-thalassemias are a group of hereditary diseases caused by more than 300 mutations of the adult β-globin gene, leading to low or absent production of adult hemoglobin (HbA) (1-3). Together with sickle cell anemia (SCA), thalassemia syndromes are among the most impactful diseases in developing countries, in which the lack of genetic counselling and prenatal diagnosis have contributed to the maintenance of a very high frequency in the population. The management of β-thalassemia patients is mostly based on blood transfusion, chelation therapy and, alternatively, on bone marrow transplantation (2). Recently, novel therapeutic options have been explored, such as gene therapy (3) and fetal hemoglobin (HbF) induction (4). Despite the fact that these approaches are promising, they are at present still under deep experimental development and limited to a low number of clinical trials (2-4). With respect to gene therapy for β-thalassemia significant progresses are expected, also considering fundamental insights into globin switching and new technology developments which might have a strong impact on novel gene-therapy approaches (3). A robust information is however available regarding the management of β-thalassemia, i.e., that patients exhibiting high levels of endogenous HbF might exhibit a milder clinical status, as in the case of hereditary persistence of fetal hemoglobin (HPFH) (4). In this context, one of the most exciting strategies recently proposed for hereditary diseases, including β-thalassemia, is genome editing using a variety of strongly validated approaches. Among these strategies, the clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 nuclease system (5-7), in which a single guide RNA (sgRNA) directs the Cas9 nuclease for site-specific cleavage, is considered the most efficient
Bioethics and Freedom of Scientific Research in Gene Therapy and Stem Cell Biology
No full textSeveral projects and research fields are expected to strongly contribute to solving therapeutic or diagnostic issues by providing new technological solutions for molecular healthcare. One example, related to diagnosis, is the innovation in the field of development of workflows, methods and devices for analysis (even multiplexed) of biomarkers (RNA and proteins in live cells) and high throughput molecular diagnostics for personalized therapy. On the other hand, novel technologies related to regenerative medicine have brought great promises for the treatment of a large number of human disease, but also relevant ethical issues that are expected to limit significantly the freedom of research in several states, several of them belonging to the European Union. This is especially related with the research on human embryonic stem cells (hESCs), which is expected to bring important novelty in developing approaches in regenerative medicine. On the other hand, the ban of patenting invention based on embryonic stem cells appears to limit this approach, since scientists working in stem cell medicine will not be able to deliver clinical benefits without the involvement of biological industry, which must have patent protection as an incentive to be involved in this research activity. Limitation of the freedom of research in this field should be considered
Therapeutic Relevance of Inducing Autophagy in β-Thalassemia
No full textThe beta-thalassemias are inherited genetic disorders affecting the hematopoietic system. In beta-thalassemias, more than 350 mutations of the adult beta-globin gene cause the low or absent production of adult hemoglobin (HbA). A clinical parameter affecting the physiology of erythroid cells is the excess of free alpha-globin. Possible experimental strategies for a reduction in excess free alpha-globin chains in beta-thalassemia are CRISPR-Cas9-based genome editing of the beta-globin gene, forcing "de novo" HbA production and fetal hemoglobin (HbF) induction. In addition, a reduction in excess free alpha-globin chains in beta-thalassemia can be achieved by induction of the autophagic process. This process is regulated by the Unc-51-like kinase 1 (Ulk1) gene. The interplay with the PI3K/Akt/TOR pathway, with the activity of the alpha-globin stabilizing protein (AHSP) and the involvement of microRNAs in autophagy and Ulk1 gene expression, is presented and discussed in the context of identifying novel biomarkers and potential therapeutic targets for beta-thalassemia
Gene silencing in the study of transcriptional regulation: the A beta H-J-J locus
No full textThe starting point to explain how the main biological processes occur is the understanding of the mechanisms which control the gene expression regulation. One of the mechanisms of transcriptional regulation is operating at the promoter level. in this context, the identification and characterization of transcription factors Involved on promoter binding and regulation appears to be very important. Silencing strategies are useful methods to study the function of transcription factors (TFs) as promoter regulators, and they include: 1) decoy and 2) RNA interference (RNAi) approaches. This article describes a successful application of these two methods in the study of transcriptional regulation of human A beta H-J-J locus. Transcription factor decoy (TFD) approach was employed in order to determine the role of Sp1-related proteins; RNAi approach for upstream stimulatory transcription factor (USF) depletion was used to study the role of this factor on P1 promoter directed expression
Structural characterization of promoter sequences of the gene coding human PKI55 protein, a protein kinase C inhibitor
No full textThe PKI55 protein was identified in our laboratory as specific protein kinase C inhibitor. We previously demonstrated that PKI55 is poorly translated in vivo and acts promoting PKC degradation and establishing a feedback loop of inhibition. However, our understanding of mechanisms by which the expression of PKI55 is regulated, is limited. In the present work we investigated the mRNA expression of PKI55 in human tissues by Northern blotting and RT- PCR, demonstrating that it is highly expressed in brain tissue. Moreover, since the computational analysis of the gene promoter region showed two sites (Box 1 and Box 2) similar to consensus sequences for AP1 and GAGA-factors, we investigated their ability to bind to these proteins. Electrophoretic Mobility Shift Assays showed that GAGA-factors preferentially interacted with Box 2, while AP-1 elements linked preferentially Box 1 sequence. We suggest that the interaction of these transcription factors with Box 1 and Box 2 could regulate the transcription of the PKI55 gene and, consequently, the expression of PKC
SPR-based identification of microRNAs
No full textMicroRNAs (miRNAs) are small non-coding endogenous RNAs acting as post-transcriptional regulators of gene expression in eukaryotes, that play important roles in homeostatic cell processes such as development, proliferation and apoptosis. Recently, the deregulation of miRNA levels has been linked to the pathogenesis of several human diseases, including cancer initiation and progression, making them useful as prognostic and diagnostic biomarkers. Among the novel detection strategies required to identify and quantify specific miRNAs in biological samples, ultrasensitive and specific methods based on Surface Plasmon Resonance are under development in order to overcome some pitfalls of conventional miRNA diagnostic techniques
Preliminary results and a theoretical perspective of co‐treatment using a miR‐93‐5p mimic and aged garlic extract to inhibit the expression of the pro‐inflammatory interleukin‐8 gene
No full textThe coronavirus disease-19 (COVID-19) pandemic has been a very significant health issue in the period between 2020 and 2023, forcing research to characterize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequences and to develop novel therapeutic approaches. Interleukin-6 (IL-6) and IL-8 are considered significant therapeutic targets for COVID-19 and emerging evidence has suggested that microRNAs (miRNAs/miRs) serve a key role in regulating these genes. MiRNAs are short, 19-25 nucleotides in length, non-coding RNAs that regulate gene expression at the post-transcriptional level through the sequence-selective recognition of the 3'-untranslated region (3'-UTR) of the regulated mRNAs, eventually repressing translation, commonly, via mRNA degradation. For example, among several miRNAs involved in the regulation of the COVID-19 'cytokine storm', miR-93-5p can inhibit IL-8 gene expression by directly targeting the 3'-UTR of IL-8 mRNA. In addition, miR-93-5p can regulate Toll-like receptor-4 (TLR4) and interleukin-1 receptor-associated kinase 4 (IRAK4) expression, thus affecting the nuclear factor-kappa B (NF-kappa B) pathway and the expression of NF-kappa B-regulated genes, such as IL-6, IL-1 beta and other hyper-expressed genes during the COVID-19 'cytokine storm'. In the present study, the results provided preliminary evidence suggesting that the miR-93-5p-based miRNA therapeutics could be combined with the anti-inflammatory aged garlic extract (AGE) to more effectively inhibit IL-8 gene expression. The human bronchial epithelial IB3-1 cell line was employed as experimental model system. IB3-1 cells were stimulated with the BNT162b2 COVID-19 vaccine and transfected with pre-miR-93-5p in the absence or in the presence of AGE, to verify the inhibitory effects on the BNT162b2-induced expression of the IL-8 gene. The accumulation of IL-8 mRNA was assessed by RT-qPCR; the release of IL-8 protein was determined by Bio-Plex assay. In addition, the possible applications of TLR4/NF-kappa B inhibitory agents (such as miR-93-5p and AGE) for treating human pathologies at a hyperinflammatory state, such as COVID-19, cystic fibrosis and other respiratory diseases, were summarized
- …
