Comparative analysis of lipid Nanoparticle-Mediated delivery of CRISPR-Cas9 RNP versus mRNA/sgRNA for gene editing in vitro and in vivo.

The discovery that the bacterial defense mechanism, CRISPR-Cas9, can be reprogrammed as a gene editing tool has revolutionized the field of gene editing. CRISPR-Cas9 can introduce a double-strand break at a specific targeted site within the genome. Subsequent intracellular repair mechanisms repair the double strand break that can either lead to gene knock-out (via the non-homologous end-joining pathway) or specific gene correction in the presence of a DNA template via homology-directed repair. With the latter, pathological mutations can be cut out and repaired. Advances are being made to utilize CRISPR-Cas9 in patients by incorporating its components into non-viral delivery vehicles that will protect them from premature degradation and deliver them to the targeted tissues. Herein, CRISPR-Cas9 can be delivered in the form of three different cargos: plasmid DNA, RNA or a ribonucleoprotein complex (RNP). We and others have recently shown that Cas9 RNP can be efficiently formulated in lipid-nanoparticles (LNP) leading to functional delivery in vitro. In this study, we compared LNP encapsulating the mRNA Cas9, sgRNA and HDR template against LNP containing Cas9-RNP and HDR template. Former showed smaller particle sizes, better protection against degrading enzymes and higher gene editing efficiencies on both reporter HEK293T cells and HEPA 1-6 cells in in vitro assays. Both formulations were additionally tested in female Ai9 mice on biodistribution and gene editing efficiency after systemic administration. LNP delivering mRNA Cas9 were retained mainly in the liver, with LNP delivering Cas9-RNPs additionally found in the spleen and lungs. Finally, gene editing in mice could only be concluded for LNP delivering mRNA Cas9 and sgRNA. These LNPs resulted in 60 % gene knock-out in hepatocytes. Delivery of mRNA Cas9 as cargo format was thereby concluded to surpass Cas9-RNP for application of CRISPR-Cas9 for gene editing in vitro and in vivo.

Vitamin C facilitates direct cardiac reprogramming by inhibiting reactive oxygen species.

BACKGROUND: After myocardial infarction, the lost myocardium is replaced by fibrotic tissue, eventually progressively leading to myocardial dysfunction. Direct reprogramming of fibroblasts into cardiomyocytes via the forced overexpression of cardiac transcription factors Gata4, Mef2c, and Tbx5 (GMT) offers a promising strategy for cardiac repair. The limited reprogramming efficiency of this approach, however, remains a significant challenge. METHODS: We screened seven factors capable of improving direct cardiac reprogramming of both mice and human fibroblasts by evaluating small molecules known to be involved in cardiomyocyte differentiation or promoting human-induced pluripotent stem cell reprogramming. RESULTS: We found that vitamin C (VitC) significantly increased cardiac reprogramming efficiency when added to GMT-overexpressing fibroblasts from human and mice in 2D and 3D model. We observed a significant increase in reactive oxygen species (ROS) generation in human and mice fibroblasts upon Doxy induction, and ROS generation was subsequently reduced upon VitC treatment, associated with increased reprogramming efficiency. However, upon treatment with dehydroascorbic acid, a structural analog of VitC but lacking antioxidant properties, no difference in reprogramming efficiency was observed, suggesting that the effect of VitC in enhancing cardiac reprogramming is partly dependent of its antioxidant properties. CONCLUSIONS: Our findings demonstrate that VitC supplementation significantly enhances the efficiency of cardiac reprogramming, partially by suppressing ROS production in the presence of GMT.

A lipid nanoparticle platform for mRNA delivery through repurposing of cationic amphiphilic drugs.

Since the recent clinical approval of siRNA-based drugs and COVID-19 mRNA vaccines, the potential of RNA therapeutics for patient healthcare has become widely accepted. Lipid nanoparticles (LNPs) are currently the most advanced nanocarriers for RNA packaging and delivery. Nevertheless, the intracellular delivery efficiency of state-of-the-art LNPs remains relatively low and safety and immunogenicity concerns with synthetic lipid components persist, altogether rationalizing the exploration of alternative LNP compositions. In addition, there is an interest in exploiting LNP technology for simultaneous encapsulation of small molecule drugs and RNA in a single nanocarrier. Here, we describe how well-known tricyclic cationic amphiphilic drugs (CADs) can be repurposed as both structural and functional components of lipid-based NPs for mRNA formulation, further referred to as CADosomes. We demonstrate that selected CADs, such as tricyclic antidepressants and antihistamines, self-assemble with the widely-used helper lipid DOPE to form cationic lipid vesicles for subsequent mRNA complexation and delivery, without the need for prior lipophilic derivatization. Selected CADosomes enabled efficient mRNA delivery in various in vitro cell models, including easy-to-transfect cancer cells (e.g. human cervical carcinoma HeLa cell line) as well as hard-to-transfect primary cells (e.g. primary bovine corneal epithelial cells), outperforming commercially available cationic liposomes and state-of-the-art LNPs. In addition, using the antidepressant nortriptyline as a model compound, we show that CADs can maintain their pharmacological activity upon CADosome incorporation. Furthermore, in vivo proof-of-concept was obtained, demonstrating CADosome-mediated mRNA delivery in the corneal epithelial cells of rabbit eyes, which could pave the way for future applications in ophthalmology. Based on our results, the co-formulation of CADs, helper lipids and mRNA into lipid-based nanocarriers is proposed as a versatile and straightforward approach for the rational development of drug combination therapies.

Delivery of modified mRNA to damaged myocardium by systemic administration of lipid nanoparticles.

Lipid Nanoparticles (LNPs) are a promising drug delivery vehicle for clinical siRNA delivery. Modified mRNA (modRNA) has recently gained great attention as a therapeutic molecule in cardiac regeneration. However, for mRNA to be functional, it must first reach the diseased myocardium, enter the target cell, escape from the endosomal compartment into the cytosol and be translated into a functional protein. However, it is unknown if LNPs can effectively deliver mRNA, which is much larger than siRNA, to the ischemic myocardium. Here, we evaluated the ability of LNPs to deliver mRNA to the myocardium upon ischemia-reperfusion injury functionally. By exploring the bio-distribution of fluorescently labeled LNPs, we observed that, upon reperfusion, LNPs accumulated in the infarct area of the heart. Subsequently, the functional delivery of modRNA was evaluated by the administration of firefly luciferase encoding modRNA. Concomitantly, a significant increase in firefly luciferase expression was observed in the heart upon myocardial reperfusion when compared to sham-operated animals. To characterize the targeted cells within the myocardium, we injected LNPs loaded with Cre modRNA into Cre-reporter mice. Upon LNP infusion, Tdtomato+ cells, derived from Cre mediated recombination, were observed in the infarct region as well as the epicardial layer upon LNP infusion. Within the infarct area, most targeted cells were cardiac fibroblasts but also some cardiomyocytes and macrophages were found. Although the expression levels were low compared to LNP-modRNA delivery into the liver, our data show the ability of LNPs to functionally deliver modRNA therapeutics to the damaged myocardium, which holds great promise for modRNA-based cardiac therapies.

CRISPR/Cas9-mediated knockin of human factor IX into swine factor IX locus effectively alleviates bleeding in hemophilia B pigs.

Hemophilia B is an X-linked recessive bleeding disorder caused by abnormalities in the coagulation factor IX gene. Without prophylactic treatment, patients experience frequent spontaneous bleeding episodes. Well-characterized animal models are valuable for determining the pathobiology of the disease and testing novel therapeutic innovations. Here, we generated a porcine model of hemophilia B using a combination of CRISPR/Cas9 and somatic cell nuclear transfer. Moreover, we tested the possibility of hemophilia B therapy by gene insertion. Frequent spontaneous joint bleeding episodes that occurred in hemophilia B pigs allowed a thorough investigation of the pathological process of hemophilic arthropathy. In contrast to the hemophilia B pigs, which showed a severe bleeding tendency and joint damage, the transgenic pigs carrying human coagulation factor IX exhibited a partial improvement of bleeding. In summary, this study not only offers a translational hemophilia B model for exploring the pathological process of hemophilic arthropathy but also provides a possibility for the permanent correction of hemophilia in the future by genome editing in situ.

Repairing the heart: State-of the art delivery strategies for biological therapeutics.

Myocardial infarction (MI) is one of the leading causes of mortality worldwide. It is caused by an acute imbalance between oxygen supply and demand in the myocardium, usually caused by an obstruction in the coronary arteries. The conventional therapy is based on the application of (a combination of) anti-thrombotics, reperfusion strategies to open the occluded artery, stents and bypass surgery. However, numerous patients cannot fully recover after these interventions. In this context, new therapeutic methods are explored. Three decades ago, the first biologicals were tested to improve cardiac regeneration. Angiogenic proteins gained popularity as potential therapeutics. This is not straightforward as proteins are delicate molecules that in order to have a reasonably long time of activity need to be stabilized and released in a controlled fashion requiring advanced delivery systems. To ensure long-term expression, DNA vectors-encoding for therapeutic proteins have been developed. Here, the nuclear membrane proved to be a formidable barrier for efficient expression. Moreover, the development of delivery systems that can ensure entry in the target cell, and also correct intracellular trafficking towards the nucleus are essential. The recent introduction of mRNA as a therapeutic entity has provided an attractive intermediate: prolonged but transient expression from a cytoplasmic site of action. However, protection of the sensitive mRNA and correct delivery within the cell remains a challenge. This review focuses on the application of synthetic delivery systems that target the myocardium to stimulate cardiac repair using proteins, DNA or RNA.

Genetically modified pigs are protected from classical swine fever virus.

Classical swine fever (CSF) caused by classical swine fever virus (CSFV) is one of the most detrimental diseases, and leads to significant economic losses in the swine industry. Despite efforts by many government authorities to stamp out the disease from national pig populations, the disease remains widespread. Here, antiviral small hairpin RNAs (shRNAs) were selected and then inserted at the porcine Rosa26 (pRosa26) locus via a CRISPR/Cas9-mediated knock-in strategy. Finally, anti-CSFV transgenic (TG) pigs were produced by somatic nuclear transfer (SCNT). Notably, in vitro and in vivo viral challenge assays further demonstrated that these TG pigs could effectively limit the replication of CSFV and reduce CSFV-associated clinical signs and mortality, and disease resistance could be stably transmitted to the F1-generation. Altogether, our work demonstrated that RNA interference (RNAi) technology combining CRISPR/Cas9 technology offered the possibility to produce TG animal with improved resistance to viral infection. The use of these TG pigs can reduce CSF-related economic losses and this antiviral strategy may be useful for future antiviral research.

Truncated C-terminus of fibrillin-1 induces Marfanoid-progeroid-lipodystrophy (MPL) syndrome in rabbit.

Various clinical differences have been observed between patients with the FBN1 gene mutation and those with the classical Marfan phenotype. Although FBN1 knockout (KO) or dominant-negative mutant mice are widely used as an animal model for Marfan syndrome (MFS), these mice cannot recapitulate the genotype/phenotype relationship of Marfanoid-progeroid-lipodystrophy (MPL) syndrome, which is caused by a mutation in the C-terminus of fibrillin-1, the penultimate exon of the FBN1 gene. Here, we describe the generation of a rabbit MPL model with C-terminal truncation of fibrillin-1 using a CRISPR/Cas9 system. FBN1 heterozygous (FBN1 Het) rabbits faithfully recapitulated the phenotypes of MFS, including muscle wasting and impaired connective tissue, ocular syndrome and aortic dilation. Moreover, skin symptoms, lipodystrophy, growth retardation and dysglycemia were also seen in these FBN1 Het rabbits, and have not been reported in other animal models. In conclusion, this novel rabbit model mimics the histopathological changes and functional defects of MPL syndrome, and could become a valuable model for studies of pathogenesis and drug screening for MPL syndrome.

Metformin ameliorates the progression of atherosclerosis via suppressing macrophage infiltration and inflammatory responses in rabbits.

AIMS: The present study aimed to investigate the possible effects of metformin on the progression of atherosclerosis in a rabbit model. MAIN METHODS: Rabbits were randomly divided into three groups (n = 10): the control (Ctrl) group (fed with a chow diet), and two experimental groups, the AS group and the Met group (both received an atherogenic diet). After 2 weeks of acclimatization, the rabbits in the AS and Met groups were given a placebo and metformin, respectively, daily by gavage for 10 weeks. Plasma lipids and inflammatory cytokines were measured. The aorta was isolated for histological and immunohistochemical analysis. In vitro, human umbilical vein endothelial cells (HUVECs) were treated with metformin, and monocyte adhesion and adhesion molecule expression were measured. KEY FINDINGS: Metformin reduced plasma inflammatory cytokine levels but did not alter lipid content. Compared with that in the AS group, the atherosclerosis burden in the Met group was significantly decreased. The lesional macrophage content was reduced, but the lesional collagen content was not affected in the metformin-treated rabbits, compared with the corresponding levels in the non-treated controls. Furthermore, the aortic mRNA expression levels of adhesion molecules and inflammatory cytokines in the Met group were also significantly reduced compared with those in the AS group. Metformin treatment reduced monocyte adhesion to endothelial cells (ECs) and adhesion molecule expression, and inhibited rabbit monocyte differentiation into macrophages and the macrophage inflammatory response. SIGNIFICANCE: Our results suggest that metformin impeded the progression of atherosclerosis, possibly by suppressing macrophage infiltration and inflammatory responses.

Resveratrol suppresses lipoprotein-associated phospholipase A2 expression by reducing oxidative stress in macrophages and animal models.

SCOPE: Resveratrol is a naturally occurring polyphenolic compound with known cardioprotective, anti-inflammatory, and antioxidant properties. Lipoprotein-associated phospholipase A2 (Lp-PLA2 ) is associated with the risk of cardiovascular disease. Here, we investigated the effects of resveratrol on Lp-PLA2 expression in vitro and in vivo and explored the underlying mechanisms. METHODS AND RESULTS: Human monocytic cells (THP-1) were induced to differentiate into macrophages for an in vitro experimental model. Resveratrol suppressed Lp-PLA2 expression and reduced inflammation; lipopolysaccharide (LPS, 1 µg/mL), tumor necrosis factor-α (TNF-α, 10 ng/mL) and reactive oxygen species (ROS) were employed to stimulate an increase in Lp-PLA2 expression and ROS levels, and the stimulation was inhibited by resveratrol (50 µM) and other antioxidants. The inhibition of resveratrol was inversed partially by sirtuin 1 (SIRT1) inhibitors (Nicotinamide, 1-10 mM) (p<0.05). Next, a chronic inflammation mouse model induced by a HFD (high fat diet) supplemented with resveratrol 100 mg/kg/day orally for 12 weeks, resulted in resveratrol-induced decreases in the Lp-PLA2 levels in the plasma and liver and increases in the superoxide dismutase 2 (SOD2) expression in the liver (p<0.05). CONCLUSION: Based on our results, the protective effects of resveratrol on cardiovascular events may be related to its ability to suppress Lp-PLA2 expression.