Brain-targeted intranasal delivery of protein-based gene therapy for treatment of ischemic stroke.

Gene therapy using a protein-based CRISPR system in the brain has practical limitations due to current delivery systems, especially in the presence of arterial occlusion. To overcome these obstacles and improve stability, we designed a system for intranasal administration of gene therapy for the treatment of ischemic stroke. Methods: Nanoparticles containing the protein-based CRISPR/dCas9 system targeting Sirt1 were delivered intranasally to the brain in a mouse model of ischemic stroke. The CRISPR/dCas9 system was encapsulated with calcium phosphate (CaP) nanoparticles to prevent them from being degraded. They were then conjugated with ß-hydroxybutyrates (bHb) to target monocarboxylic acid transporter 1 (MCT1) in nasal epithelial cells to facilitate their transfer into the brain. Results: Human nasal epithelial cells were shown to uptake and transfer nanoparticles to human brain endothelial cells with high efficiency in vitro. The intranasal administration of the dCas9/CaP/PEI-PEG-bHb nanoparticles in mice effectively upregulated the target gene, Sirt1, in the brain, decreased cerebral edema and increased survival after permanent middle cerebral artery occlusion. Additionally, we observed no significant in vivo toxicity associated with intranasal administration of the nanoparticles, highlighting the safety of this approach. Conclusion: This study demonstrates that the proposed protein-based CRISPR-dCas9 system targeting neuroprotective genes in general, and SIRT1 in particular, can be a potential novel therapy for acute ischemic stroke.

Somatic Variants in SVIL in Cerebral Aneurysms.

Background and Objectives: While somatic mutations have been well-studied in cancer, their roles in other complex traits are much less understood. Our goal is to identify somatic variants that may contribute to the formation of saccular cerebral aneurysms. Methods: We performed whole-exome sequencing on aneurysm tissues and paired peripheral blood. RNA sequencing and the CRISPR/Cas9 system were then used to perform functional validation of our results. Results: Somatic variants involved in supervillin (SVIL) or its regulation were found in 17% of aneurysm tissues. In the presence of a mutation in the SVIL gene, the expression level of SVIL was downregulated in the aneurysm tissue compared with normal control vessels. Downstream signaling pathways that were induced by knockdown of SVIL via the CRISPR/Cas9 system in vascular smooth muscle cells (vSMCs) were determined by evaluating changes in gene expression and protein kinase phosphorylation. We found that SVIL regulated the phenotypic modulation of vSMCs to the synthetic phenotype via Krüppel-like factor 4 and platelet-derived growth factor and affected cell migration of vSMCs via the RhoA/ROCK pathway. Discussion: We propose that somatic variants form a novel mechanism for the development of cerebral aneurysms. Specifically, somatic variants in SVIL result in the phenotypic modulation of vSMCs, which increases the susceptibility to aneurysm formation. This finding suggests a new avenue for the therapeutic intervention and prevention of cerebral aneurysms.

Auranofin, an Anti-rheumatic Gold Drug, Aggravates the Radiation-Induced Acute Intestinal Injury in Mice.

Pelvic and abdominal radiotherapy plays an important role in eradication of malignant cells; however, it also results in slight intestinal injury. The apoptosis of cells in the intestinal epithelium is a primary pathological factor that initiates radiation-induced intestinal injury. Auranofin, a gold-containing triethylphosphine, was approved for the treatment of rheumatoid arthritis, and its therapeutic application has been expanded to a number of other diseases, such as parasitic infections, neurodegenerative disorders, AIDS, and bacterial infections. Recently, a treatment strategy combining the use of auranofin and ionizing radiation aimed at increasing the radiosensitivity of cancer cells was proposed for improving the control of local cancers. In this study, we evaluated the effect of auranofin on the radiosensitivity of intestinal epithelial cells. The treatment with a combination of 1 µM auranofin and 5 Gy ionizing radiation showed clear additive effects on caspase 3 cleavage and apoptotic DNA fragmentation in IEC-6 cells, and auranofin administration significantly aggravated the radiation-induced intestinal injury in mice. Auranofin treatment also resulted in the activation of the unfolded protein response and in the inhibition of thioredoxin reductase, which is a key component of the cellular antioxidant system. Pre-treatment with N-acetyl cysteine, a well-known scavenger of reactive oxygen species, but not with a chemical chaperone, which inhibits endoplasmic reticulum stress and the ensuing unfolded protein response, significantly reduced the radiosensitizing effects of auranofin in the IEC-6 cells. In addition, transfection of IEC-6 cells with a small interfering RNA targeted against thioredoxin reductase significantly enhanced the radiosensitivity of these cells. These results suggest that auranofin-induced radiosensitization of intestinal epithelial cells is mediated through oxidative stress caused by the deregulation of thioredoxin redox system, and auranofin treatment can be an independent risk factor for the development of acute pelvic radiation disease.

Lecithin nano-liposomal particle as a CRISPR/Cas9 complex delivery system for treating type 2 diabetes.

BACKGROUND: Protein-based Cas9 in vivo gene editing therapeutics have practical limitations owing to their instability and low efficacy. To overcome these obstacles and improve stability, we designed a nanocarrier primarily consisting of lecithin that can efficiently target liver disease and encapsulate complexes of Cas9 with a single-stranded guide RNA (sgRNA) ribonucleoprotein (Cas9-RNP) through polymer fusion self-assembly. RESULTS: In this study, we optimized an sgRNA sequence specifically for dipeptidyl peptidase-4 gene (DPP-4) to modulate the function of glucagon-like peptide 1. We then injected our nanocarrier Cas9-RNP complexes directly into type 2 diabetes mellitus (T2DM) db/db mice, which disrupted the expression of DPP-4 gene in T2DM mice with remarkable efficacy. The decline in DPP-4 enzyme activity was also accompanied by normalized blood glucose levels, insulin response, and reduced liver and kidney damage. These outcomes were found to be similar to those of sitagliptin, the current chemical DPP-4 inhibition therapy drug which requires recurrent doses. CONCLUSIONS: Our results demonstrate that a nano-liposomal carrier system with therapeutic Cas9-RNP has great potential as a platform to improve genomic editing therapies for human liver diseases.