Ground state naïve pluripotent stem cells and CRISPR/Cas9 gene correction for β-thalassemia

The 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

Therapy for Cystic Fibrosis Caused by Nonsense Mutations

Nonsense mutations cover about 10% of cystic fibrosis (CF) patients and generate premature termination codons (PTCs) leading to premature translational termination and causing the synthesis of truncated non-functional or partially functional CFTR (cystic fibrosis transmembrane conductance regulator) protein. The read-through approach is the suppression of translation terminations at PTCs and it has been developed as a therapeutic strategy to restore full-length protein using aminoglycoside antibiotics or PTC124. Phenotypic consequences of PTCs can be exacerbated by the nonsense-mediated mRNA decay (NMD) pathway, which detects and degrades mRNA containing PTC. Therefore, modulation of NMD is also of interest as a potential target for suppression therapy. Not all PTCs are susceptible to the read-through treatment alone, especially where the nonsense mutations are combined with other CFTR mutations. For example, many CF patients present the highly frequent F508del CF mutation, causing an alteration of the cell membrane positioning of the CFTR channel. Pharmacological correctors that rescue the trafficking of F508del CFTR may overcome this defect. A combined administration of correctors/potentiators, read-through molecules, and/or NMD inhibitors, depending on the genotype of the CF patients, could be the basis for the design of a personalized therapeutic approach

Induction by TNF- α

We have developed a microencapsulation procedure for the entrapment and manipulation of IB3-1 cystic fibrosis cells. The applied method is based on generation of monodisperse droplets by a vibrational nozzle. Different experimental parameters were analyzed, including frequency and amplitude of vibration, polymer pumping rate and distance between the nozzle and the gelling bath. We have found that the microencapsulation procedure does not alter the viability of the encapsulated IB3-1 cells. The encapsulated IB3-1 cells were characterized in term of secretomic profile, analyzing the culture medium by Bio-Plex strategy. The experiments demonstrated that most of the analyzed proteins, were secreted both by the free and encapsulated cells, even if in a different extent. In order to determine the biotechnological applications of this procedure, we determined whether encapsulated IB3-1 cells could be induced to pro-inflammatory responses, after treatment with TNF-α. In this experimental set-up, encapsulated and free IB3-1 cells were treated with TNF-α, thereafter the culture media from both cell populations were collected. As expected, TNF-α induced a sharp increase in the secretion of interleukins, chemokines and growth factors. Of great interest was the evidence that induction of interleukin-6 and interleukin-8 occurs also by encapsulated IB3-1 cells

Expression of miR-210 during erythroid differentiation and induction of γ-globin gene expression

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A Rational Approach to Drug Repositioning in β-thalassemia: Induction of Fetal Hemoglobin by Established Drugs

Drug repositioning and the relevance of orphan drug designation for β-thalassemia is reviewed. Drug repositioning and similar terms ('drug repurposing', 'drug reprofiling', 'drug redirecting', 'drug rescue', 'drug re-tasking' and/or 'drug rediscovery') have gained great attention, especially in the field or rare diseases (RDs), and represent relevant novel drug development strategies to be considered together with the 'off-label' use of pharmaceutical products under clinical trial regimen. The most significant advantage of drug repositioning over traditional drug development is that the repositioned drug has already passed a significant number of short- and long-term toxicity tests, as well as it has already undergone pharmacokinetic and pharmacodynamic (PK/PD) studies. The established safety of repositioned drugs is known to significantly reduce the probability of project failure. Furthermore, development of repurposed drugs can shorten much of the time needed to bring a drug to market. Finally, patent filing of repurposed drugs is expected to catch the attention of pharmaceutical industries interested in the development of therapeutic protocols for RDs. Repurposed molecules that could be proposed as potential drugs for β-thalassemia, will be reported, with some of the most solid examples, including sirolimus (rapamycin) that recently has been tested in a pilot clinical trial

Applications to cancer research of "lab-on-a-chip" devices based on dielectrophoresis (DEP).

The recent development of advanced analytical and bioseparation methodologies based on microarrays and biosensors is one of the strategic objectives of the so-called post-genomic. In this field, the development of microfabricated devices could bring new opportunities in several application fields, such as predictive oncology, diagnostics and anti-tumor drug research. The so called "Laboratory-on-a-chip technology", involving miniaturisation of analytical procedures, is expected to enable highly complex laboratory testing to move from the central laboratory into non-laboratory settings. The main advantages of Lab-on-a-chip devices are integration of multiple steps of different analytical procedures, large variety of applications, sub-microliter consumption of reagents and samples, and portability. One of the requirement for new generation Lab-on-a-chip devices is the possibility to be independent from additional preparative/analytical instruments. Ideally, Lab-on-a-chip devices should be able to perform with high efficiency and reproducibility both actuating and sensing procedures. In this review, we discuss applications of dielectrophoretic(DEP)-based Lab-on-a-chip devices to cancer research. The theory of dielectrophoresis as well as the description of several devices, based on spiral-shaped, parallel and arrayed electrodes are here presented. In addition, in this review we describe manipulation of cancer cells using advanced DEP-based Lab-on-a-chip devices in the absence of fluid flow and with the integration of both actuating and sensing procedures

BCL11A mRNA targeting by miR-210: A possible network regulating γ-globin gene expression

The involvement of microRNAs in the control of repressors of human g-globin gene transcription has been firmly demonstrated, as described for the miR-486-3p mediated down-regulation of BCL11A. On the other hand, we have reported that miR-210 is involved in erythroid differentiation and, possibly, in γ-globin gene up-regulation. In the present study, we have identified the coding sequence of BCL11A as a possible target of miR-210. The following results sustain this hypothesis: (a) interactions between miR-210 and the miR-210 BCL11A site were demonstrated by SPR-based biomolecular interaction analysis (BIA); (b) the miR-210 site of BCL11A is conserved through molecular evolution; (c) forced expression of miR-210 leads to decrease of BCL11A-XL and increase of γ-globin mRNA content in erythroid cells, including erythroid precursors isolated from β-thalassemia patients. Our study suggests that the coding mRNA sequence of BCL11A can be targeted by miR-210. In addition to the theoretical point of view, these data are of interest from the applied point of view, supporting a novel strategy to inhibit BCL11A by mimicking miR-210 functions, accordingly with the concept supported by several papers and patent applications that inhibition of BCL11A is an efficient strategy for fetal hemoglobin induction in the treatment of β-thalassemia

Bergamot (Citrus bergamia Risso) fruit extracts and identified components alter expression of interleukin 8 gene in cystic fibrosis bronchial epithelial cell lines

<p>Abstract</p> <p>Background</p> <p>Cystic fibrosis (CF) airway pathology is a fatal, autosomal, recessive genetic disease characterized by extensive lung inflammation. After induction by TNF-α, elevated concentrations of several pro-inflammatory cytokines (i.e. IL-6, IL-1β) and chemokines (i.e. IL-8) are released from airway epithelial cells. In order to reduce the excessive inflammatory response in the airways of CF patients, new therapies have been developed and in this respect, medicinal plant extracts have been studied. In this article we have investigated the possible use of bergamot extracts (<it>Citrus bergamia </it>Risso) and their identified components to alter the expression of IL-8 associated with the cystic fibrosis airway pathology.</p> <p>Methods</p> <p>The extracts were chemically characterized by ¹H-NMR (nuclear magnetic resonance), GC-FID (gas chromatography-flame ionization detector), GC-MS (gas chromatography-mass spectrometry) and HPLC (high pressure liquid chromatography). Both bergamot extracts and main detected chemical constituents were assayed for their biological activity measuring (a) cytokines and chemokines in culture supernatants released from cystic fibrosis IB3-1 cells treated with TNF-α by Bio-Plex cytokine assay; (b) accumulation of IL-8 mRNA by real-time PCR.</p> <p>Results</p> <p>The extracts obtained from bergamot (<it>Citrus bergamia </it>Risso) epicarps contain components displaying an inhibitory activity on IL-8. Particularly, the most active molecules were bergapten and citropten. These effects have been confirmed by analyzing mRNA levels and protein release in the CF cellular models IB3-1 and CuFi-1 induced with TNF-α or exposed to heat-inactivated <it>Pseudomonas aeruginosa</it>.</p> <p>Conclusions</p> <p>These obtained results clearly indicate that bergapten and citropten are strong inhibitors of IL-8 expression and could be proposed for further studies to verify possible anti-inflammatory properties to reduce lung inflammation in CF patients.</p

Postnatal and non-invasive prenatal detection of β-thalassemia mutations based on Taqman genotyping assays

The β-thalassemias are genetic disorder caused by more than 200 mutations in the β-globin gene, resulting in a total (β0) or partial (β+) deficit of the globin chain synthesis. The most frequent Mediterranean mutations for β-thalassemia are: β039, β+ VSI-110, β+IVSI-6 and β0IVSI-1. Several molecular techniques for the detection of point mutations have been developed based on the amplification of the DNA target by polymerase chain reaction (PCR), but they could be labor-intensive and technically demanding. On the contrary, Taq- Man® genotyping assays are a simple, sensitive and versatile method suitable for the single nucleotide polymorphism (SNP) genotyping affecting the human β-globin gene. Four Taq- Man® genotyping assays for the most common β-thalassemia mutations present in the Mediterranean area were designed and validated for the genotype characterization of genomic DNA extracted from 94 subjects comprising 25 healthy donors, 33 healthy carriers and 36 β- thalassemia patients. In addition, 15 specimens at late gestation (21-39 gestational weeks) and 11 at early gestation (5-18 gestational weeks) were collected from pregnant women, and circulating cell-free fetal DNAs were extracted and analyzed with these four genotyping assays. We developed four simple, inexpensive and versatile genotyping assays for the postnatal and prenatal identification of the thalassemia mutations β039, β+IVSI-110, β+IVSI-6, β0IVSI-1. These genotyping assays are able to detect paternally inherited point mutations in the fetus and could be efficiently employed for non-invasive prenatal diagnosis of β-globin gene mutations, starting from the 9th gestational week

Evaluation of S-RBD and high specificity ACE-2-binding antibodies on SARS-CoV-2 patients after six months from infection

The antibody response to SARS-CoV-2 has not yet fully defined, but the availability of sensitive and specific serological assays is crucial to observe the presence of specific antibodies against the human receptor binding domain (S-RBD) and high specificity ACE-2-binding antibodies or neutralizing antibodies (NT) in response to vaccines. Indeed, these peculiar antibodies should prevent viral interaction between RBD and AngiotensinConverting Enzyme 2 (ACE2) receptor, located on surface of host cells. In this study, 72 samples from 37 hospitalized COVID-19 patients and 35 not-hospitalized patients were analyzed longitudinally. The detection of SRBD and NT antibodies was carried out using CLIA tests. Hospitalized patients showed elevated serum levels of S-RBD (97.22%) and NT (77.78%) antibodies, differently, not-hospitalized, who were paucisymptomatic or asymptomatic patients, showed lower serum levels of SRBD (65.71%) and NT (38.14%) antibodies. The results suggest that the NT serum level is strongly related to disease severity (p &lt; 0.001) and to the serum level of S-RBD antibodies (p &lt; 0.0001)