Characterization of stability, safety and immunogenicity of the mRNA lipid nanoparticle vaccine Iribovax® against COVID-19 in nonhuman primates.

mRNA-lipid nanoparticle (mRNA-LNP) vaccines have proved their efficacy, versatility and unprecedented manufacturing speed during the COVID-19 pandemic. Here we report on the physicochemical properties, thermostability, immunogenicity, and protective efficacy of the nucleoside-modified mRNA-LNP vaccine candidate Iribovax® (also called SNEG2c). Injection of BALB/c mice, rabbits and nonhuman primates with two doses of SNEG2c induced production of high-titers of SARS-CoV-2 spike-specific and receptor-binding domain (RBD)-neutralizing antibodies in immunized animals. In addition to the strong humoral response, SNEG2c elicited substantial Th1-biased T-cell response. Sera from rhesus macaques immunized with a low dose of the vaccine showed robust spike-specific antibody titers 3-24× as high as those in convalescent sera from a panel of COVID-19 patients and 50% virus neutralization geometric mean titer of 1024 against SARS-CoV-2. Strikingly, immunization with SNEG2c completely cleared infectious SARS-CoV-2 from the upper and lower respiratory tracts of challenged macaques and protected them from viral-induced lung and trachea lesions. In contrast, the non-vaccinated macaques developed moderate to severe pulmonary pathology after the viral challenge. We present the results of repeat-dose and local tolerance toxicity and thermostability studies showing how the physicochemical properties of the mRNA-LNPs change over time and demonstrating that SNEG2 is safe, well tolerated and stable for long-term. These results support the planned human trials of SNEG2c.

A novel and easy to prepare azo-based bioreductive linker and its application in hypoxia-sensitive cationic liposomal doxorubicin: Synthesis, characterization, in vitro and in vivo studies in mice bearing C26 tumor.

This study designed and synthesized a cost-effective azo-based hypoxia-sensitive linker (AHSL) using commercially accessible, inexpensive raw materials and simple methods to apply in cationic nanoliposomes. Then, AHSL was post-inserted into the cationic liposome (Cat-lip), and PEG-Azo-Cat-lip was prepared and characterized using DLS. The decrease in the zeta-potential of formulation from + 18.4 mV for Cat-lip to + 6.1 mV and the increase in the size of the PEG-Azo-Cat-lip indicated the successful post insertion of AHSL into the liposomes. The Doxorubicin (Dox) release study showed that PEGylation results in a more stable PEG-Azo-Cat-lip than the Cat-lip. The increased cytotoxicity of the PEG-Azo-Cat-lip in the hypoxic condition also indicated the cleavage of the AHSL in the hypoxic environment. In vivo biodistribution using animal imaging has shown higher tumor accumulation of the MPEG-Azo-Cat-lip than Cat-lip during the 120 h of the study. The results of anti-tumor activities and biosafety of the formulations also showed the higher efficiency of the MPEG-Azo-Cat-lip compared with the Cat-lip. The results of this study indicated the antitumor efficacy of this hypoxia-sensitive which merits further investigation.

Transposase-CRISPR mediated targeted integration (TransCRISTI) in the human genome.

Various methods have been used in targeted gene knock-in applications. CRISPR-based knock-in strategies based on homology-independent repair pathways such as CRISPR HITI have been shown to possess the best efficiency for gene knock-in in mammalian cells. However, these methods suffer from the probability of plasmid backbone insertion at the target site. On the other hand, studies trying to combine the targeting ability of the Cas9 molecule and the excision/integration capacity of the PB transposase have shown random integrations. In this study, we introduce a new homology-independent knock-in strategy, Transposase-CRISPR mediated Targeted Integration (TransCRISTI), that exploits a fusion of Cas9 nuclease and a double mutant piggyBac transposase. In isogenic mammalian cell lines, we show that the TransCRISTI method demonstrates higher efficiency (72%) for site-specific insertions than the CRISPR HITI (44%) strategy. Application of the TransCRISTI method resulted in site-directed integration in 4.13% and 3.69% of the initially transfected population in the human AAVS1and PML loci, respectively, while the CRISPR HITI strategy resulted in site-directed integration in the PML locus in only 0.6% of cells. We also observed lower off-target and random insertions in the TransCRISTI group than the CRISPR HITI group. The TransCRISTI technology represents a great potential for the accurate and high-efficiency knock-in of the desired transposable elements into the predetermined genomic locations.

From DNA break repair pathways to CRISPR/Cas-mediated gene knock-in methods.

Various DNA breaks created via programmable CRISPR/Cas9 nuclease activity results in different intracellular DNA break repair pathways. Based on the cellular repair pathways, CRISPR-based gene knock-in methods can be categorized into two major strategies: 1) Homology-independent strategies which are targeted insertion events based on non-homologous end joining, and 2) Homology-dependent strategies which are targeted insertion events based on the homology-directed repair. This review elaborates on various gene knock-in methods in mammalian cells using the CRISPR/Cas9 system and in sync with DNA-break repair pathways. Gene knock-in methods are applied in functional genomics and gene therapy. To compensate or correct genetic defects, different CRISPR-based gene knock-in strategies can be used. Thus, researchers need to make a conscious decision about the most suitable knock-in method. For a successful gene-targeted insertion, some determinant factors should be considered like cell cycle, dominant DNA repair pathway, size of insertions, and donor properties. In this review, different aspects of each gene knock-in strategy are discussed to provide a framework for choosing the most appropriate gene knock-in method in different applications.

Fabrication of biopolymer based nanocomposite wound dressing: evaluation of wound healing properties and wound microbial load.

The aim of this study is to introduce natural-based polymers, chitosan and starch, to design a remedial nanocomposite, comprising of cerium oxide nanoparticles and silver nanoparticles, to investigate their effects in accelerating wound healing and in wound microbial load. Cerium oxide nanoparticles synthesized in starch solution added to the colloidal dispersion of synthesized silver nanoparticles in chitosan to make a three-component nanomaterial. Mice were anaesthetized and two parallel full-thickness round wounds were excised under aseptic conditions with the help of sterile dermal biopsy punch. Furthermore, effects of silver-chitosan and silver-cerium-chitosan nanocomposite had evaluated on rate of wound closure and collagen density and on microbial load of wound in full-thickness model. Results showed that both silver chitosan and silver-cerium-chitosan had significant impact on acceleration of wound closure and collagen content and on reduction of wound microbial load in comparison with control group, which was, received no treatments. However, the silver-cerium-chitosan nanocomposite is more potent than silver-chitosan group and control group in wound closure. The wound healing effects of silver-cerium-chitosan nanocomposite are due to unique features of its three components and this nanocomposite promises impressive remedies for clinical application.

Evaluation of Antimicrobial and Healing Activities of Frog Skin on Guinea Pigs Wounds.

BACKGROUND: Frog skin secretions have potentials against a wide spectrum of bacteria. Also, frog skin compositions have healing properties. OBJECTIVES: The aim of this study was to investigate the antibacterial potentials along with healing properties of frog skin Rana ridibunda, a species which thoroughly lives in Iran marshes, as a biological dressing on wounds. MATERIALS AND METHODS: In this study, excisional wounds, dressed with frog skin, were compared between experimental and control groups of guinea pigs. In the experimental groups, wounds were dressed with the dermal (FS) and epidermal (RFS) sides of fresh frog R. ridibunda skin, while only usual cotton gauze covered the wounds of the control group. Furthermore, microbial samples were taken on different days (0, 3, 5, and 7 days post injury) to count the number of the colony-forming units. Additionally, the microbial penetration test was performed on frog skin and then the progression of wound closure was evaluated. RESULTS: In the microbial studies, the bacterial load considerably declined in the wounds treated with FS and RFS compared with the control wounds. On day 7 post injury, the numbers of the colony-forming units for the FS, RFS, and control groups were 6.75, 105, and 375, respectively. In the penetration test, fresh frog skin showed to be a bacterial resistant dressing. The results revealed that the rate of wound closure in the experimental groups significantly was accelerated in comparison with that in the control group. CONCLUSIONS: Our results demonstrated the antimicrobial properties of frog skin as a wound dressing, which has antimicrobial effects per se. This biological dressing shows promise as an effective biological wound dressing insofar as not only is it capable of resisting microbes and accelerating wound healing but also it is cost-effective and easy to use.

Plant-mediated biosynthesis of silver nanoparticles using Prosopis farcta extract and its antibacterial properties.

"Green" synthesis of metal nanoparticles has become a promising synthetic strategy in nanoscience and nanotechnology in recent years. In this work, silver nanoparticles (Ag-NPs) were synthesized from extract of Prosopis farcta at room temperature. Formation of Ag-NPs at 1 mM concentration of AgNO3 gave spherical shape nanoparticles with mean diameter about 10.8 nm. The formation of nanoparticle was confirmed by the surface Plasmon resonance (SPR) band illustrated in UV-vis spectrophotometer. The morphology and size of the Ag-NPs were determined using high magnification transmission electron microscopy (TEM). The crystalline structure of obtained nanoparticles was investigated using the powder X-ray diffraction (PXRD) pattern. In addition, these green synthesized Ag-NPs were found to show higher antibacterial activity against multi drug resistant clinical isolates.

Pharmaceutical application of frog skin on full-thickness skin wound healing in mice.

CONTEXT: It has been proved that fresh frog skin is efficient in the wound healing process. OBJECTIVE: The purpose of study is to introduce a formulation of frog skin powder for evaluation of wound repair where fresh frog skin is not available. MATERIALS AND METHODS: Rana ridibunda (Ranidae) skins were lyophilized, and a powder was prepared. The powder (0.0005 g) was then mixed with ointment (0.0065 g) for treating each wound. Formulation was used on full-thickness wounds on mice (FO group) and compared to positive and negative controls. In order to study the wound healing process, wound contraction, inflammation, number of fibroblast cells, neovascularization and collagen density were evaluated on days 2, 4 and 6 following the injury. Moreover, CFU measurement was performed for the evaluation of wound contamination. RESULTS: Acceleration in wound contraction in the FO group compared to control groups was significant (p < 0.001) on days 4 and 6. Results showed that FO treatment considerably decreased inflammatory cells during the study. On day 4, FO treatment was significantly effective in increasing the number of fibroblast cells and collagen density (p < 0.01 and p < 0.05, respectively). On day 6 the number of fibroblast cells (p < 0.001), collagen density (p < 0.05) and neovascularization (p < 0.05), were higher in the FO group than the control groups. Results of CFU measurement demonstrated significant reduction of wound contamination in FO treated wounds on days 2 (p < 0.05) and 4 (p < 0.01). DISCUSSION AND CONCLUSION: Our findings indicated that the pharmaceutical form of frog skin used in this study has considerable healing and antibacterial effects on wounds.

Topical effects of frog "Rana ridibunda" skin secretions on wound healing and reduction of wound microbial load.

ETHNOPHARMACOLOGICAL RELEVANCE: Study of the interrelationships between human and the animals in their environment has always been a subject of interest and caused discoveries of the animal applications in medicine. From the latest century, these remedies called back in traditional medicine of Vietnam and South America and frog skin was used as a biological dressing and had good effects in healing wounds. Also, frog skin secretions have wound healing properties and reduce inflammation. In this study we applied these secretions in the form of ointment to investigate their healing activities. MATERIALS AND METHODS: Skin secretions were extracted from Rana ridibunda to evaluate their effects on wound healing in mice. Secretion used as raw extract (RE) and ultrafiltrated extract, using a membrane with cutoff 10kDa as under 10kDa (U10E), was administrated as ointment every 48h on wound site. Control group was left without any treatment and also there was other group treated with ointment (O group) alone. On 2, 4 and 6 days post injury, animals were euthanized and images were taken for wound closure evaluation. Then wound locations were removed for histological assays. Also wound microbial load was examined. Observational parameters including wound closure and wound microbiology in experimental groups compared with the control and O groups have been studied. RESULTS: The results showed U10E group has better effects than RE, O and control groups. Histological parameters, including numbers of inflammatory and fibroblast cells and amount of collagen fibers, neovascularization, as well, represented greater degree of wound healing in U10E group compared with RE, O, and control groups. CONCLUSIONS: Our results showed that frog skin secretions were significantly effective in promoting wound healing process. The U10E extract from the frog R. ridibunda possesses a potent accelerating wound healing effect that promises good potential for clinical application in wound care. Further studies will be required to characterize special molecules encompassing healing properties.