Base editing by nucleotide deaminases linked to programmable DNA-binding proteins represents a promising approach to permanently remedy blood disorders, although its application in engrafting ...hematopoietic stem cells (HSCs) remains unexplored. In this study, we purified A3A (N57Q)-BE3 base editor for ribonucleoprotein (RNP) electroporation of human-peripheral-blood-mobilized CD34
hematopoietic stem and progenitor cells (HSPCs). We observed frequent on-target cytosine base edits at the BCL11A erythroid enhancer at +58 with few indels. Fetal hemoglobin (HbF) induction in erythroid progeny after base editing or nuclease editing was similar. A single therapeutic base edit of the BCL11A enhancer prevented sickling and ameliorated globin chain imbalance in erythroid progeny from sickle cell disease and β-thalassemia patient-derived HSPCs, respectively. Moreover, efficient multiplex editing could be achieved with combined disruption of the BCL11A erythroid enhancer and correction of the HBB -28A>G promoter mutation. Finally, base edits could be produced in multilineage-repopulating self-renewing human HSCs with high frequency as assayed in primary and secondary recipient animals resulting in potent HbF induction in vivo. Together, these results demonstrate the potential of RNP base editing of human HSPCs as a feasible alternative to nuclease editing for HSC-targeted therapeutic genome modification.
Although base editors are useful tools for precise genome editing, current base editors can only convert either adenines or cytosines. We developed a dual adenine and cytosine base editor (A&C-BEmax) ...by fusing both deaminases with a Cas9 nickase to achieve C-to-T and A-to-G conversions at the same target site. Compared to single base editors, A&C-BEmax's activity on adenines is slightly reduced, whereas activity on cytosines is higher and RNA off-target activity is substantially decreased.
Re-expression of the paralogous γ-globin genes (HBG1/2) could be a universal strategy to ameliorate the severe β-globin disorders sickle cell disease (SCD) and β-thalassemia by induction of fetal ...hemoglobin (HbF, α
γ
)
. Previously, we and others have shown that core sequences at the BCL11A erythroid enhancer are required for repression of HbF in adult-stage erythroid cells but are dispensable in non-erythroid cells
. CRISPR-Cas9-mediated gene modification has demonstrated variable efficiency, specificity, and persistence in hematopoietic stem cells (HSCs). Here, we demonstrate that Cas9:sgRNA ribonucleoprotein (RNP)-mediated cleavage within a GATA1 binding site at the +58 BCL11A erythroid enhancer results in highly penetrant disruption of this motif, reduction of BCL11A expression, and induction of fetal γ-globin. We optimize conditions for selection-free on-target editing in patient-derived HSCs as a nearly complete reaction lacking detectable genotoxicity or deleterious impact on stem cell function. HSCs preferentially undergo non-homologous compared with microhomology-mediated end joining repair. Erythroid progeny of edited engrafting SCD HSCs express therapeutic levels of HbF and resist sickling, while those from patients with β-thalassemia show restored globin chain balance. Non-homologous end joining repair-based BCL11A enhancer editing approaching complete allelic disruption in HSCs is a practicable therapeutic strategy to produce durable HbF induction.
ChatGPT has promising applications in health care, but potential ethical issues need to be addressed proactively to prevent harm. ChatGPT presents potential ethical challenges from legal, humanistic, ...algorithmic, and informational perspectives. Legal ethics concerns arise from the unclear allocation of responsibility when patient harm occurs and from potential breaches of patient privacy due to data collection. Clear rules and legal boundaries are needed to properly allocate liability and protect users. Humanistic ethics concerns arise from the potential disruption of the physician-patient relationship, humanistic care, and issues of integrity. Overreliance on artificial intelligence (AI) can undermine compassion and erode trust. Transparency and disclosure of AI-generated content are critical to maintaining integrity. Algorithmic ethics raise concerns about algorithmic bias, responsibility, transparency and explainability, as well as validation and evaluation. Information ethics include data bias, validity, and effectiveness. Biased training data can lead to biased output, and overreliance on ChatGPT can reduce patient adherence and encourage self-diagnosis. Ensuring the accuracy, reliability, and validity of ChatGPT-generated content requires rigorous validation and ongoing updates based on clinical practice. To navigate the evolving ethical landscape of AI, AI in health care must adhere to the strictest ethical standards. Through comprehensive ethical guidelines, health care professionals can ensure the responsible use of ChatGPT, promote accurate and reliable information exchange, protect patient privacy, and empower patients to make informed decisions about their health care.
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•The poly(ionic liquid)/PVA hydrogel dressing does not release antibacterial agents.•This hydrogel dressing exhibits effective antibacterial ability and good mechanical property.•The ...mouse osteoblasts growth on the surface of hydrogel certified its excellent biocompatibility.•This hydrogel dressing can promote the reconstruction of intact epidermis.
Antibacterial wound dressings play an important role in wound healing and infection treatment. However, traditional antibacterial wound dressings uncontrollably release antibiotics or silver ions through passive diffusion. The overuse of antibiotics and silver ions may cause drug resistance, side effects, and argyrism, thereby hindering the healing process and creating other health hazards. To overcome these shortcomings, this paper reports an easy approach to synthesize non-releasing antimicrobial Poly(ionic liquid)/PVA hydrogel dressing with high-strength through chemical polymerization and physical cross-linking. The hydrogel dressing exhibited effective antibacterial ability against bacteria (E. coli, S. aureus and B. subtilis), fungus (C. albicans), and mold (Asp. niger, Asp. oryzae, and Rhizopus). Furthermore, in a murine model, the hydrogel dressing efficiently accelerated cutaneous wound healing. After 15 days of healing process, histological tests indicated that this hydrogel dressing can promote the reconstruction of intact epidermis faster than the control. Therefore, this Poly(ionic liquid)/PVA hydrogel has potential as an antibacterial wound-healing dressing.
Designing heterogeneous solid surface frustrated Lewis pair (ssFLP) catalyst for hydrogenation is a new challenge in catalysis and no research has been reported on the construction of ssFLP on ...boehmite (AlOOH) surfaces up to now as far as we know. Herein, AlOOH with a layer structure is prepared and it is found that the Lewis basic O
site (one H removed from OH) and an adjacent Lewis acidic unsaturated Al site (Al
.) proximal to a surface OH
(OH vacancy) on AlOOH layers could form the ssFLP. The layered structure of AlOOH and its abundant OH defects over the surface result in a high concentration of O
/Al
. FLPs, which are conducive to highly efficient hydrogen activation for hydrogenation of olefins and alkynes with low H-H bond dissociates activation energy of 0.16 eV under mild conditions (T = 80°C and P(H
) = 2.0 MPa). This work develops a new kind of hydrogenation catalyst and provides a new perspective for creating solid surface FLP.
The CRISPR-Cas9 system has been employed to generate mutant alleles in a range of different organisms. However, so far there have not been reports of use of this system for efficient correction of a ...genetic disease. Here we show that mice with a dominant mutation in Crygc gene that causes cataracts could be rescued by coinjection into zygotes of Cas9 mRNA and a single-guide RNA (sgRNA) targeting the mutant allele. Correction occurred via homology-directed repair (HDR) based on an exogenously supplied oligonucleotide or the endogenous WT allele, with only rare evidence of off-target modifications. The resulting mice were fertile and able to transmit the corrected allele to their progeny. Thus, our study provides proof of principle for use of the CRISPR-Cas9 system to correct genetic disease.
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•A dominant cataract-causing mutation in the Crygc gene is corrected using CRISPR-Cas9•Genetic correction via HDR uses information from the endogenous WT allele•Genetic correction can also occur using information from an exogenous oligo•The rescued mice can transmit the corrected allele to their progeny
The authors show genetic rescue of a dominant cataract-causing mutation in mice using an injection of CRISPR-Cas9 and a guide RNA into zygotes.
To investigate clinicopathological variables influencing overall survival, overall recurrence, and post-recurrence survival (PRS) in patients who experienced curative-intent surgical resection of ...stage I non-small-cell lung cancer (NSCLC).
We investigated a series of 1387 patients with stage I NSCLC who underwent surgical resection from 2008 to 2015. The effect clinicopathological factors on death, recurrence, and PRS were evaluated by Kaplan-Meier estimates and cox regression analysis.
Among the 1387 stage I patients, 301 (21.7%) experienced recurrence. The 5-year cumulative incidence of recurrence (CIR) for all patients was 20.2% and median PRS was 25.5 months. The older age (P = 0.036), p-stage IB (P = 0.001), sublobar resection(P<0.001), histology subtype (P<0.001), and lymphovascular invasion (LVI) (P = 0.042) were significantly associated with worse overall survival. Among 301 recurrent patients, univariable analysis indicated that p-stage IB (versus IA) (P<0.001), LVI (P<0.001) and visceral pleural invasion (VPI) (P<0.001) were remarkably correlated with the higher incidence of recurrence. Taking the effect of clinicopathological variables on PRS into consideration, smoking history (P = 0.043), non-adenocarcinoma (P = 0.013), high architectural grade of LUAD (P = 0.019), EGFR wild status (P = 0.002), bone metastasis (P =0.040) and brain metastasis (P = 0.042) were substantially related with poorer PRS. Multivariate analysis demonstrated that high architectural grade of LUAD (P = 0.008), brain metastasis (P = 0.010) and bone metastasis (P = 0.043) were independently associated with PRS.
In patients with resected stage I NSCLC, the older age, p-stage IB (versus IA), sublobar resection, histology subtype, and LVI were significantly associated with worse overall survival. P-stage IB (versus IA), LVI, and VPI were significantly correlated with the higher incidence of recurrence. High architectural grade of LUAD, brain metastasis and bone metastasis were independent risk factors with PRS.
Spermatogonial stem cells （SSCs） can produce numerous male gametes after transplantation into recipient testes, presenting a valuable approach for gene therapy and continuous production of ...gene-modified animals. However, successful genetic manipulation of SSCs has been limited, partially due to complexity and low efficiency of currently available genetic editing techniques. Here, we show that efficient genetic modifications can be introduced into SSCs using the CRISPR-Cas9 system. We used the CRISPR-Cas9 system to mutate an EGFP transgene or the endogenous Crygc gene in SCCs. The mutated SSCs underwent spermatogenesis after transplantation into the seminiferous tubules of infertile mouse testes. Round spermatids were generated and, after injection into mature oocytes, supported the production of heterozygous offspring displaying the corresponding mutant phenotypes. Furthermore, a disease-causing mutation in Crygc （Crygc-/-） that pre-existed in SSCs could be readily repaired by CRISPR-Cas9-induced nonhomologous end joining （NHEJ） or homology-directed repair （HDR）, resulting in SSC lines carrying the corrected gene with no evidence of off-target modifications as shown by whole-genome sequencing. Fertilization using round spermatids generated from these lines gave rise to offspring with the corrected phenotype at an efficiency of 100%. Our results demonstrate efficient gene editing in mouse SSCs by the CRISPR-Cas9 system, and provide the proof of principle of curing a genetic disease via gene correction in SSCs.
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