[Advances in gene therapy for ß-thalassemia and hemophilia based on the CRISPR/Cas9 technology].

Thalassemia and hemophilia are common inherited blood disorders caused by genetic abnormalities. These diseases are difficult to cure and can be inherited to the next generation, causing severe family and social burden. The emergence of gene therapy provides a new treatment for genetic diseases. However, since its first clinical trial in 1990, the development of gene therapy has not been as optimistic in the past three decades as one could hope. The development of gene-editing technology, particularly the third generation gene-editing technology CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9), has given hope in such therapeutic approach for having advantages in high editing efficiency, simple operation, and low cost. Gene editing-mediated gene therapy has thus received increasing attention from the biomedical community. It has shown promises for the treatment of inherited blood disorders, such as thalassemia and hemophilia. This paper reviews the fundamental research progress of gene therapy for ß-thalassemia and hemophilia based on CRISPR/Cas9 technology in the past six years. It also summarizes the CRISPR/Cas9-based clinical trials of gene therapy. The problems and possible solutions to this technology for gene therapy are also discussed, thereby providing a reference for the research on gene therapy of inherited blood disorders based on CRISPR/Cas9 technology.

Integration-free methods for generating induced pluripotent stem cells.

Induced pluripotent stem (iPS) cells can be generated from mouse or human fibroblasts by exogenous expression of four factors, Oct4, Sox2, Klf4 and c-Myc, and hold great potential for transplantation therapies and regenerative medicine. However, use of retroviral vectors during iPS cell generation has limited the technique's clinical application due to the potential risks resulting from genome integration of transgenes, including insertional mutations and altered differentiation potentials of the target cells, which may lead to pathologies such as tumorigenesis. Here we review recent progress in generating safer transgene-free or integration-free iPS cells, including the use of non-integrating vectors, excision of vectors after integration, DNA-free delivery of factors and chemical induction of pluripotency.

Donor-host mitochondrial compatibility improves efficiency of bovine somatic cell nuclear transfer.

BACKGROUND: The interaction between the karyoplast and cytoplast plays an important role in the efficiency of somatic cell nuclear transfer (SCNT), but the underlying mechanism remains unclear. It is generally accepted that in nuclear transfer embryos, the reprogramming of gene expression is induced by epigenetic mechanisms and does not involve modifications of DNA sequences. In cattle, oocytes with various mitochondrial DNA (mtDNA) haplotypes usually have different ATP content and can further affect the efficiency of in vitro production of embryos. As mtDNA comes from the recipient oocyte during SCNT and is regulated by genes in the donor nucleus, it is a perfect model to investigate the interaction between donor nuclei and host oocytes in SCNT. RESULTS: We investigated whether the in vitro development of reconstructed bovine embryos produced by SCNT would be influenced by mtDNA haplotype compatibility between the oocytes and donor cells. Embryos from homotype A-A or B-B showed significantly higher developmental ability at blastocyst stages than the heterotype A-B or B-A combinations. Post-implantation development ability, pregnancy rate up to day 90 of gestation, as well as percent of term births were higher in the homotype SCNT groups than in the heterotype groups. In addition, homotype and heterotype SCNT embryos showed different methylation patterns of histone 3-lysine 9 (H3K9) genome-wide and at pluripotency-related genes (Oct-4, Sox-2, Nanog). CONCLUSION: Both histone and DNA methylation show that homotype SCNT blastocysts have a more successful epigenetic asymmetry pattern than heterotype SCNT blastocysts, which indicates more complete nuclear reprogramming. This may result from variability in their epigenetic patterns and responses to nuclear reprogramming. This suggests that the compatibility of mtDNA haplotypes between donor cells and host oocytes can significantly affect the developmental competence of reconstructed embryos in SCNT, and may include an epigenetic mechanism.

Improved efficiency of bovine somatic cell nuclear transfer by optimizing operational procedures.

To improve bovine somatic cell nuclear transfer (SCNT) efficiency, we studied various aspects to optimize the experimental procedures. Firstly, donor cells were treated with pronase, which resulted in a higher fusion rate than that of cells without the pronase treatment (78.3 vs. 53.9%). Secondly, when fused embryos were activated either by chemical (ionomycin + cyclohemixide (CHX)) or electrical + CHX stimulation, the cleavage and blastocyst formation rates were comparable amongst these treatment groups (P>0.05); however, mortality following electrical + CHX activation was significantly higher than that observed with the chemical activation, regardless of the pronase treatment (P<0.05). Finally, we compared the culture conditions of the reconstructed embryos using ACM medium plus mouse embryonic fibroblasts (MEF) vs. B2 medium plus granulose cells (GC), and the results clearly demonstrated that the former culture conditions led to a higher blastocyst rate, 90-day pregnancy rate, and newborn rate, than that observed for culture in B2 medium plus GC (46.7 vs. 34.7%, 36.1 vs. 9.6% and 25.9 vs. 5.8% for the blastocyst, pregnancy and newborn rates, respectively). In summary, the efficiency of bovine SCNT can be greatly improved using optimized operational procedures, including treating the donor cells with pronase, activation of fused embryos by ionomycin + CHX and the culture of the reconstructed embryos in ACM + MEF media.

[Two vital transcriptional factors Oct-4 and Nanog to keep the pluripotency and self-renewal of stem cells and related regulation network].

Oct-4 and Nanog are two critical transcriptional factors to keep pluripotency and self-renewal of stem cells in vivo and in vitro, and they usually express only in pluripotent cells and not in differentiated cells. They bind to the regulatory regions of targeted gene and often interact with other transcriptional factors and extracellular signal path components, such as Sox-2, FoxD3, LIF and BMP in specific tissues or developmental stages. So that all of these constitute a transcriptional crosstalk, and finally determine the cells destiny: keeping pluripotency or turning to differentiation.