Therapeutic Effects of Hematopoietic Stem Cell Derived From Gene-Edited Mice on ß654-Thalassemia.

ß-thalassemia is an inherited blood disease caused by reduced or inadequate ß-globin synthesis due to ß-globin gene mutation. Our previous study developed a gene-edited mice model (ß654-ER mice) by CRISPR/Cas9-mediated genome editing, targeting both the ßIVS2-654 (C > T) mutation site and the 3' splicing acceptor site at 579 and corrected abnormal ß-globin mRNA splicing in the ß654-thalassemia mice. Herein, we further explored the therapeutic effect of the hematopoietic stem cells (HSCs) from ß654-ER mice on ß-thalassemia by consecutive HSC transplantation. The results indicated that HSC transplantation derived from gene-edited mice can significantly improve the survival rate of mice after lethal radiation doses and effectively achieve hematopoietic reconstruction and long-term hematopoiesis. Clinical symptoms, including hematologic parameters and tissue pathology of transplanted recipients, were significantly improved compared to the non-transplanted ß654 mice. The therapeutic effect of gene-edited HSC transplantation demonstrated no significant difference in hematological parameters and tissue pathology compared with wild-type mouse-derived HSCs. Our data revealed that HSC transplantation from gene-edited mice completely recovered the ß-thalassemia phenotype. Our study systematically investigated the therapeutic effect of HSCs derived from ß654-ER mice on ß-thalassemia and further confirmed the efficacy of our gene-editing approach. Altogether, it provided a reference and primary experimental data for the clinical usage of such gene-edited HSCs in the future.

Correction of RNA splicing defect in ß654-thalassemia mice using CRISPR/Cas9 gene-editing technology.

ß654-thalassemia is a prominent Chinese subtype of b-thalassemia, representing 17% of all cases of ß-thalassemia in China. The molecular mechanism underlying this subtype involves the IVS-2-654 C→T mutation leading to aberrant ß-globin RNA splicing. This results in an additional 73-nucleotide exon between exons 2 and 3 and leads to a severe thalassemia syndrome. Herein, we explored a CRISPR/Cas9 genome editing approach to eliminate the additional 73- nucleotide by targeting both the IVS-2-654 C→T and a cryptic acceptor splice site at IVS-2-579 in order to correct aberrant b-globin RNA splicing and ameliorate the clinical ß-thalassemia syndrome in ß654 mice. Gene-edited mice were generated by microinjection of sgRNA and Cas9 mRNA into one-cell embryos of ß654 or control mice: 83.3% of live-born mice were gene-edited, 70% of which produced correctly spliced RNA. No off-target events were observed. The clinical symptoms, including hematologic parameters and tissue pathology of all of the edited ß654 founders and their offspring were significantly improved compared to those of the non-edited ß654 mice, consistent with the restoration of wild-type b-globin RNA expression. Notably, the survival rate of gene-edited heterozygous ß654 mice increased significantly, and liveborn homozygous ß654 mice were observed. Our study demonstrated a new and effective gene-editing approach that may provide groundwork for the exploration of ß654-thalassemia therapy in the future.

miR-124-3p Inhibits Microglial Secondary Inflammation After Basal Ganglia Hemorrhage by Targeting TRAF6 and Repressing the Activation of NLRP3 Inflammasome.

Objectives: Intracerebral hemorrhage (ICH) represents a serious central nervous system emergency with high morbidity and mortality, and the basal ganglia is the most commonly affected brain region. Differentially expressed microRNAs (miRs) have recently been highlighted to serve as potential diagnostic biomarkers and therapeutic targets for ICH. This study investigated the mechanism of miR-124-3p in microglial secondary inflammation after ICH. Methods: In this study, 48 patients with primary basal ganglia ICH and 48 healthy volunteers were selected and venous blood was collected from all patients on the second morning of admission (within 24 h of stroke onset). The expression of miR-124-3p in serum was detected by RT-qPCR. Three months after ICH, the patients were assessed by modified Rankin Scale (mRS), and the correlation between miR-124-3p expression and mRS score was analyzed by Pearson. The inflammatory response of microglia was induced by lipopolysaccharide (LPS) to establish the cell model of microglial inflammation. miR-124-3p expression patterns were detected in the serum of ICH patients and healthy volunteers, normal microglia, and LPS-induced microglia. The miR-124-3p mimic was transfected into LPS-induced microglia, followed by measurement of the inflammatory factors, apoptosis rate, and cell viability. The target gene of miR-124-3p was predicted and verified. The expression patterns of tumor necrosis factor receptor-associated factor 6 (TRAF6) were detected. pcDNA3.1 and pcDNA3.1-TRAF6 were transfected into LPS-induced HMC3 cells, and nucleotide-binding oligomerization domain-like receptor (NLR) pyrin domain-containing 3 (NLRP3) expression patterns were determined. Lastly, the effects of TRAF6 overexpression on apoptosis, cell viability, and inflammation in HMC3 cells were measured. Results: miR-124-3p was downregulated in the serum of basal ganglia ICH patients and LPS-induced microglia, and miR-124-3p expression was negatively correlated with mRS. Overexpression of miR-124-3p reduced the inflammatory factors and apoptosis rate and promoted cell activity in LPS-induced microglia. miR-124-3p was found to target TRAF6. Overexpression of TRAF6 enhanced the expression of NLRP3 inflammasome, inflammatory factors and apoptosis rate, and reduced cell viability. Conclusion: Our findings indicate that miR-124-3p repressed the activation of NLRP3 inflammasome by targeting TRAF6, thus inhibiting microglial secondary inflammation after ICH in basal ganglia.

The Amorphous Quercetin/ Hydroxypropylmethylcellulose Acetate Succinate Solid Dispersions Prepared by Co-Precipitation Method to Enhance Quercetin Dissolution.

HPMCAS-HF, HPMCAS-MF and HPMCAS-LF were used as carriers to prepare the amorphous solid dispersions (ASDs) of quercetin (Que) by co-precipitation. The Que ASD based on PVP K30 was prepared by solvent evaporation method. The ability of polymer to inhibit Que crystallization was evaluated. The study found the order of the ability of polymer to inhibit Que nucleation to be: HF > MF > LF > K30, and that to maintain Que supersaturation to be: HF > K30 > MF > LF. The prepared solid dispersions were characterized by IR, DSC and PXRD. Although HF was the most effective crystallization inhibitor, the release of the Que/HF ASD was poor and assigned to the carrier-controlled dissolution for the strong interactions between Que and HF. The Que/MF ASD exhibited better dissolution behavior compared to the Que/K30 ASD. The dissolution behavior of the Que ASD depended on the polymer-Que interactions and the ability of crystallization inhibition of the polymer.

EDNRA Gene rs1878406 Polymorphism is Associated With Susceptibility to Large Artery Atherosclerotic Stroke.

Objective: We performed this study to investigate whether the EDNRA gene rs1878406 C > T polymorphism is associated with risk of large artery atherosclerosis (LAA) stroke in the Chinese Han population. Methods: Genotyping of rs1878406 was performed in 1,112 LAA stroke patients and 1,192 healthy controls. Multivariate logistic regression analyses were applied to assess the effect of the rs1878406 C > T polymorphism on susceptibility to LAA stroke. Results: A significant increase of LAA stroke risk was found in the recessive model (TT vs. CC/TC, OR = 1.74, 95% CI = 1.23-2.48, p = 0.002) and co-dominant model (TC vs. CC, OR = 1.06, 95% CI = 0.89-1.27, TT vs. CC, OR = 1.79, 95% CI = 1.25-2.55, p = 0.006). However, the interaction between age and genotypes of rs1878406 was not statistically significant, and no significant interactive effect was observed between the rs1878406 C > T polymorphism and sex (p > 0.05). Conclusion: The rs1878406 C > T polymorphism is associated with increased risk of LAA stroke in the Chinese Han population.

Improved biosafety of a lentiviral vector by reducing cellular gene activation.

BACKGROUND: Lentiviral vectors (LVs) have enhancer activity and/or transcriptional read-through (EATRT) properties that can lead to the activation of adjacent genes. Consequently, patients may be at increased risk for adverse effects if such vectors are used clinically. METHODS: In the present study, we assessed the abilities of different "pro-LV"-like constructs with respect to decreasing its EATRT, including the "pro-LV" vector bearing a chimeric ΔLTR of the human foamy virus R-U5 region replaced by that of an LV (HF). RESULTS: By analyzing the EATRT of "pro-LV" constructs transfected in 293T cells, we observed that the inclusion of the first 400 bp of the chicken ß-globin locus HS4 insulator core sequence oriented in the reverse direction (C-) combined with two copies of the simian virus 40 upstream-sequence element (U) at the ΔU3 of ΔLTR region of "pro-LV" tended to shield the adjacent genomic sequences, such that the EATRT rate was lower than when either of the C- or U was included in the "pro-LV". Moreover, upon transduction, the pro-HF appears to reduce the EATRT rate in the chromosomes of 293T (by 80%) and human peripheral blood mononuclear cells (PBMCs) (by 75%) compared to when pro-LV C-U was included (with a 60% and 89% reduction in 293T and PBMCs, respectively). The HF construct had a significant reduction of viral biological titer compared tiowhen the pro-LV C-U was used in 293T cells. CONCLUSIONS: The results of the present study provide an important basis for the clinical applicability of LVs in gene and cell therapy.

MicroRNA 27b-3p Modulates SYK in Pediatric Asthma Induced by Dust Mites.

The PI3K-AKT pathway is known to regulate cytokines in dust mite-induced pediatric asthma. However, the underlying molecular steps involved are not clear. In order to clarify further the molecular steps, this study investigated the expression of certain genes and the involvement of miRNAs in the PI3K-AKT pathway, which might affect the resultant cytokine-secretion. in-vivo and in-vitro ELISA, qRT-PCR and microarrays analyses were used in this study. A down-expression of miRNA-27b-3p in dust mite induced asthma group (group D) was found by microarray analysis. This was confirmed by qRT-PCR that found the miRNA-27b-3p transcripts that regulated the expression of SYK and EGFR were also significantly decreased (p < 0.01) in group D. The transcript levels of the SYK and PI3K genes were higher, while those of EGFR were lower in the former group. Meanwhile, we found significant differences in plasma concentrations of some cytokines between the dust mite-induced asthma subjects and the healthy controls. On the other hand, this correlated with the finding that the transcripts of SYK and its downstream PI3K were decreased in HBE transfected with miRNA-27b-3p, but were increased in HBE transfected with the inhibitor in vitro. Our results indicate that the differential expression of the miRNAs in dust mite-induced pediatric asthma may regulate their target gene SYK and may have an impact on the PI3K-AKT pathway associated with the production of cytokines. These findings should add new insight into the pathogenesis of pediatric asthma.

Treatment of ß654 -thalassaemia by TALENs in a mouse model.

OBJECTIVES: This study explored whether TALENs-mediated non-homologous end joining (NHEJ) targeting the mutation site can correct the aberrant ß-globin RNA splicing, and ameliorate the ß-thalassaemia phenotype in ß654 mice. MATERIAL AND METHODS: TALENs vectors targeted to the human ß-globin gene (HBB) IVS2-654C >T mutation in a mouse model were constructed and selected to generate double heterozygous TALENs+ /ß654 mice. The gene editing and off-target effects were analysed by sequencing analysis. ß-globin expression was identified by RT-PCR and Western blot analysis. Various clinical indices including haematologic parameters and tissue pathology were examined to determine the therapeutic effect in these TALENs+ /ß654 mice. RESULTS: Sequencing analysis revealed that the HBB IVS2-654C >T point mutation was deleted in over 50% of the TALENs+ /ß654 mice tested, and off-target effects were not detected. RT-PCR and Western blot analysis confirmed the expression of normal ß-globin in TALENs+ /ß654 mice. The haematologic parameters were significantly improved as compared with their affected littermates. The proportion of nucleated cells in bone marrow was considerably decreased, splenomegaly with extramedullary haematopoiesis was reduced, and significant decreases in iron deposition were seen in spleen and liver of the TALENs+ /ß654 mice. CONCLUSION: These results suggest effective treatment of the anaemia phenotype in TALENs+ /ß654 mice following deletion of the mutation site by TALENs, demonstrating a simple and straightforward strategy for gene therapy of ß654 -thalassaemia in the future.

Chemical Changes in Grape Stem and Their Relationship to Stem Color throughout Berry Ripening in Vitis vinifera L. cv Shiraz.

Little attention has been paid to the color change or chemical compositional changes that occur in grape stems and how this correlates with the berry ripening process. Recently we have found that the change in grape peduncle color of Shiraz (Vitis vinifera) from green at veraison to predominantly brown at harvest occurs in parallel with berry ripening and as such may represent a new way of assisting in the prediction of grape maturity and harvest date. We have now investigated further the link between certain key chemical compositional changes that occur in the grape stem (peduncle and rachis) from veraison to harvest and how these attributes correlate with the observed color change in the vineyard. We report that peduncle moisture content has an excellent linear correlation with the color hue value and is negatively correlated in a strong fashion with the chlorophyll and carotenoid pigment ratio (Ca+b/Cx+c) within the peduncles. Significant differences in the moisture content, total chlorophylls (including chlorophyll a and b levels), total carotenoids, total phenolics, and the antioxidant capacity (DPPH) levels between the peduncles and rachises were found as they evolve from veraison to harvest. Finally, we have demonstrated for the first time that peduncle moisture content codevelops with the prototypical berry ripeness parameters (oBrix, pH, TA), which provides for the development of a new approach for viticulturists and winemakers to evaluate grape ripeness through peduncle moisture levels and therefore assist in harvest decision making.

Hypermethylation of the enolase gene (ENO2) in autism.

UNLABELLED: It has been hypothesized that dysregulation of brain-expressed genes is the major predisposing underlying mechanism for autism. This dysregulation may be mediated by differential methylation of CpG sites within gene promoters, which could be candidate biomarkers and used for early clinical screening of autism. A total of 131 pairs of age- and sex-matched autistic and control subjects were recruited in this study. Peripheral blood cells were analyzed. The first five pairs were randomly applied to array-based genome-wide methylation studies. A neuron-specific gene, ENO2, was found to be hypermethylated in the autistic samples. This difference was validated by bisulfite sequencing PCR (BSP). The differential expression of ENO2 gene was further analyzed with RT-qPCR and ELISA. The hypermethylation of ENO2 within the promoter region was confirmed by BSP to be present in 14.5 % (19/131) of the total of the autistic samples. The mean ENO2 RNA level in these 19 autistic samples was reduced by about 70 % relative to that in controls. The average level of ENO2 protein expression in the 19 autistic samples (15.18 ± 3.51 µg/l) was about half of that in the controls (33.86 ± 8.16 µg/l). CONCLUSION: These findings suggest that reduced ENO2 expression may be a biomarker for a subset of autistic children.

[Establishment of the methodology for quantifying lentiviral vector transcriptional read-through rate].

As an effective vehicle for bio-research and for gene therapy, Lentiviral Vector (LV) has been drawn large attention in recent years. However, transcriptional read-through limits its application. In order to understand the extend of LV read-through in chromosome, a reliable method to assess transcriptional read-through rate is needed. Here, we report the method as follows: 293T cells were transfected with the lentiviral transfer vectors which borne with two LTRs at its two ends in order to mimic the state of "proviral vectors" in chromosome. Using the primers specific for 3'U5 and 3'U3, read-through and total transcripts were reverse transcribed, respectively. These two cDNAs were quantified by realtime PCR using the primers and probe specific for 5'end of 3'U3. Read-through rate was then calculated by the division of the two. Meanwhile, read-through product of green fluorescence protein was also analyzed by Fluorescence Activated Cell Sorter. They both reciprocally proved the principal and confirmed that self-inactivated LV appeared higher read-through rate than the wild type one. The method described in this article, therefore, provides a useful technique to study how to reduce read-through rate, and improve the bio-safety of LV.

A self-deletion lentiviral vector to reduce the risk of replication-competent virus formation.

BACKGROUND: Major improvements have been made progressively on human immunodeficiency virus (HIV)-1 based lentiviral vectors to minimize the probability of replication-competent lentivirus formation. This includes the deletion of U3 promoter and the use of packaging cells, which has increased their potential for use in gene therapy and other in vivo applications. However, the risk of forming replication-competent lentiviruses remains. METHODS: We investigated the use of Cre-loxP mediation with the insertion of the transgene-expressing cassette in ΔU3 to remove additional parts of the HIV-1 backbone upon cre expression, after integration. This, leads to deletion of the packaging signal, primer binding site and Rev response element, including cre itself. RESULTS: This approach left a split truncated form of long terminal repeat flanked by a loxP and a transgene-expressing cassette in the genome, which made replication-competent lentivirus formation almost impossible. This self-deletion vector could stably express transgenes both in cell lines and transgenic mice with only modest losses of viral titer. The maximum size of the inserts was approximately 3 kb, which was sufficient for most transgenic applications. Moreover, the addition of some enhancer blocking agents downstream of the transgene could reduce the probability of transcriptional read-through in transfected 293T cells. CONCLUSIONS: Our approach could improve the biosafety of lentiviral vectors, thus improving their potential application for use in clinical trials and other in vivo applications.

Transgenic human alpha-hemoglobin stabilizing protein could partially relieve betaIVS-2-654-thalassemia syndrome in model mice.

Beta-thalassemia is an anemia caused by a relative excess of alpha-hemoglobin (alphaHb) due to absent or reduced beta-hemoglobin (betaHb) synthesis. In this study, we explore whether the introduction of alpha-hemoglobin stabilizing protein (AHSP), a chaperone protein for proper folding and stabilization of free alphaHb in red blood cells, thus aiding hemoglobin A (HbA) assembly, could relieve the pathogenic state of red blood cells in beta-thalassemia. For that, a human ahsp vector was constructed to generate transgenic human ahsp mice in a model of beta(IVS-2-654)-thalassemia by microinjecting the vector into fertilized eggs, resulting in the production of double heterozygous mice (h-ahsp(+)/beta(IVS-2-654+)). Real-time quantitative RT-PCR and Western blot analysis confirmed AHSP expression in three h-ahsp(+)/beta(IVS-2-654+) mice. Hematologic determination showed an improvement in the red blood cell indices of these h-ahsp(+)/beta(IVS-2-654+) mice. The red blood cell count and hemoglobin level were elevated to various extents as compared with their diseased siblings. A dramatic reduction in anisocytosis in the peripheral blood of h-ahsp(+)/beta(IVS-2-654+) mice was observed (16.2 +/- 4.6 vs. 30.0 +/- 5.2%). Few erythroid precursors appeared in the liver sinusoids of h-ahsp(+)/beta(IVS-2-654+) mice. Splenomegaly with extramedullary hematopoiesis was also ameliorated. Significantly, serum iron concentration was remarkably reduced as compared with that of h-ahsp(-)/beta(IVS-2-654+) mice (43.2 +/- 14.9 vs. 82.4 +/- 12.9 microM), and iron deposition in the liver was decreased in h-ahsp(+)/beta(IVS-2-654+) mice. All these results suggested amelioration of the anemia phenotype in h-ahsp(+)/beta(IVS-2-654+) mice after introduction of the ahsp gene. We therefore propose that an ahsp transgene could provide an adjuvant method for gene therapy of beta-thalassemia.

[A study of the engraftment, expansion and differentiation of human hematopoietic stem cells in goats].

OBJECTIVE: To establish a human/goat hematopoietic stem cell (HSC) xenogeneic transplant model and to probe the engraftment, expansion and differentiation of human HSC in vivo. METHODS: Human HSCs were isolated from human umbilical cord blood and 1 x 10(5) human HSCs were in utero transplanted into 50 fetal goats at the 55 - 65 the gestation days. The engraftment, expansion and differentiation of human HSCs in goats were determined by FACS analysis, PCR and PCR-Southern blot hybridization at various intervals after birth. RESULTS: Hematopoietic chimerism occurred certainly in 35 of 39 live-born recipients. On average, The proportion of human hematopoietic cells in goat blood was 1% approximately 3% and remained phenotypically stable for at least 10 months. The human hematopoietic cells circulated in goat blood expressed CD34, CD14, CD20 and glycophorin A (GPA) but did not express CD3, CD4, CD7, CD8 and CD56 or expressed them at a very low level. CONCLUSION: The number of human HSC can be effectively expanded 1 000 - 10 000 fold. Human HSCs in goats undergo a limited differentiation. Our human/goat HSC xenogeneic model provides a useful way for the investigation of HSC transplantation, expansion and differentiation in vivo.

[Molecular identification of human/goat xenogeneic model].

OBJECTIVE: To identify the human hematopoietic stem cells from the human/goat xenogeneic model with molecular techniques. METHODS: DNA and total RNA were extracted from 11 transplanted goat peripheral blood cells. Human CD(34), GPA and SRY genes were amplified with PCR in these samples, and CD(34), GPA mRNA transcripts were detected using RT-PCR in 5 and 6 goat peripheral blood cells, respectively. Southern blot analysis was performed in 8 goat DNAs to detect the human specific alpha-satellite sequence. Meanwhile FISH was also performed to detect the human cells in goat blood with a probe of human Y chromosome. RESULTS: Human CD(34) and GPA genes could be detected with PCR in all the 11 goats, and SRY gene did in 5 goats transplanted with hematopoietic stem cells derived from male human babies. Southern blot showed that human specific alpha-satellite sequence was present in 8 goats. By RT-PCR, human CD(34) mRNA was detected in 5 experimental goats, GPA mRNA was found in the other 6 experimental goats and FISH assay showed that some peripheral blood cells of the human/goat xenogeneic model were positive. CONCLUSION: Existence of human cells in the recipient goats was identified by molecular detection, which was feasible for the examination of human/goat xenogeneic models.

[The study on the stable inheritance and expression of foreign gene in transgenic mice].

Two transgenic mouse strains, in which the expression of human factor IX (hFIX) in the milk were different significantly, were bred, and the foreign gene integration as well as the content of hFIX in the milk were detected by PCR, Southern blot, FISH and ELISA, respectively. The results showed that approximately 50% offsprings were transgenic positive. Foreign gene integrated in mouse chromosomes was intact. The hFIX expression of each mouse in the same strain was different, the content of hFIX in the milk was (43.32 +/- 5.41) microgram/mL in FIX-33 transgenic strain and (1.16 +/- 0.45) microgram/mL in FIX-124 transgenic strain. Meanwhile, the hFIX gene expression between the two strains was different remarkably (P < 0.01). We conclude that the characteristics of inheritance and expression in the founder were able to be transferred to their offsprings stably.