Primary immunodeficiencies, including Wiskott-Aldrich syndrome (WAS), are a main target for genome-editing strategies using specific nucleases (SNs) because a small number of corrected hematopoietic stem cells could cure patients. In this work, we have designed various WAS gene-specific CRISPR/Cas9 systems and compared their efficiency and specificity with homodimeric and heterodimeric WAS-specific zinc finger nucleases (ZFNs), using K-562 cells as a cellular model and plasmid nucleofection or integration-deficient lentiviral vectors (IDLVs) for delivery. The various CRISPR/Cas9 and ZFN SNs showed similar efficiency when using plasmid nucleofection for delivery. However, dual IDLVs expressing ZFNs were more efficient than dual IDLVs expressing Cas9 and guide RNA or all-in-one IDLVs, expressing Cas9 and guide RNA in the same vector. The specificity of heterodimeric ZFNs and CRISPR/Cas9, measured by increments in γ-H2AX focus formation in WAS-edited cells, was similar for both, and both outperformed homodimeric ZFNs independently of the delivery system used. Interestingly, we show that delivery of SNs, using IDLVs, is more efficient and less genotoxic than plasmid nucleofection. We also show the similar behavior of heterodimeric ZFNs and CRISPR/Cas9 for homology-directed gene knock-in strategies, with 88 and 83% of the donors inserted in the WAS locus, respectively, whereas when using homodimeric ZFNs only 45% of the insertions were on target. In summary, our data indicate that CRISPR/Cas9 and heterodimeric ZFNs are both good alternatives to further develop SN-based gene therapy strategies for WAS. However, IDLV delivery of WAS-specific heterodimeric ZFNs was the best option of all systems compared in this study.
Integration-defective lentiviral vectors (IDLVs) have become an important alternative tool for gene therapy applications and basic research. Unfortunately, IDLVs show lower transgene expression as compared to their integrating counterparts. In this study, we aimed to improve the expression levels of IDLVs by inserting the IS2 element, which harbors SARs and HS4 sequences, into their LTRs (SE-IS2-IDLVs). Contrary to our expectations, the presence of the IS2 element did not abrogate epigenetic silencing by histone deacetylases. In addition, the IS2 element reduced episome levels in IDLV-transduced cells. Interestingly, despite these negative effects, SE-IS2-IDLVs outperformed SE-IDLVs in terms of percentage and expression levels of the transgene in several cell lines, including neurons, neuronal progenitor cells, and induced pluripotent stem cells. We estimated that the IS2 element enhances the transcriptional activity of IDLV LTR circles 6- to 7-fold. The final effect the IS2 element in IDLVs will greatly depend on the target cell and the balance between the negative versus the positive effects of the IS2 element in each cell type. The better performance of SE-IS2-IDLVs was not due to improved stability or differences in the proportions of 1-LTR versus 2-LTR circles but probably to a re-positioning of IS2-episomes into transcriptionally active regions.
BACKGROUND: Brain water content represents a major endpoint in studies of hepatic encephalopathy and liver failure.1 However, none of the current methods for evaluating brain water content fulfills the ideal requirements of a measuring technique, being complex, expensive, lengthy, qualitative or insensitive.2 Our AIM was to evaluate a novel protocol for measuring brain water content using a moisture analyzer in a rodent model of hyponatremia-induced brain edema. METHODS: Female Sprague-Dawley rats (248 ± 41 g bw) undergoing a 3-hour protocol for inducing hyponatremia-related brain edema (HypoNa group) were compared with a group of normonatremic rats (Control group). All rats were anesthetized with sevoflurane and mechanically ventilated. Body temperature was maintained at 37 ºC and major physiological parameters (heart rate, oxygen saturation, mean arterial pressure) were monitored. Acute hyponatremia was induced by administering desmopressin (DDAVP 1 ug/rat s.c.) and two i.p. injections of 140 mmol/L glucose solution (an injection of 12% of b.w. at the beginning and of 6% of b.w. 30 minutes later). At the end of the experiments, blood was collected from aorta, the rats were decapitated, and the brain was quickly dissected. Frontal cortex was immediately frozen and stored in liquid nitrogen. The frontal cortex was pulverized frozen with a metal mortar, and the frozen powder was placed in a moisture analyzer (MB120, Ohaus Corporation) between two glass-fiber filters for measuring water content by the wet-to-dry weight method. Dry weight (g) was determined when there was no change in 1 mg for 3 minutes. RESULTS: Compared with the Control group, rats in the HypoNa group presented lower sodium (134.0 ± 1.0 vs. 104.8 ± 1.0 mmol/L, P < 0.0001), calcium (1.27 ± 0.04 vs. 1.15 ± 0.03 mmol/L, P < 0.05), and effective osmolality (281.2 ± 2.10 vs. 229.8 ± 3.8 mOsm/L, P < 0.0001), and a trend to increased concentrations of potassium (4.10 ± 0.15 vs. 4.75 ± 0.40 mmol/L, P = 0.25) and lactate (2.23 ± 0.38 vs. 6.23 ± 2.18 mmol/L, P = 0.22) in plasma. Frontal cortex water content was higher in the HypoNa group (Control: 82.20 ± 0.74 vs. HypoNa: 85.01 ± 0.97 %, P < 0.01). Measuring time of water content using the moisture analyzer was <15 minutes per sample. CONCLUSIONS: These results suggest that the present protocol using a moisture analyzer is a convenient and sensitive method for measuring brain water content in rats.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.