It has previously been shown that engineered zinc finger nucleases (ZFNs) can be packaged into adeno-associated viruses (AAVs) and delivered intravenously into mice, non-human primates, and most recently, humans to induce highly efficient therapeutic genome editing in the liver. Lipid nanoparticles (LNPs) are synthetic delivery vehicles that enable repeat administration and are not limited by the presence of preexisting neutralizing antibodies in patients. Here, we show that mRNA encoding ZFNs formulated into LNP can enable >90% knockout of gene expression in mice by targeting the TTR or PCSK9 gene, at mRNA doses 10-fold lower than has ever been reported. Additionally, co-delivering mRNA-LNP containing ZFNs targeted to intron 1 of the ALB locus with AAV packaged with a promoterless human IDS or FIX therapeutic transgene can result in high levels of targeted integration and subsequent therapeutically relevant levels of protein expression in mice. Finally, we show repeat administration of ZFN mRNA-LNP after a single AAV donor dose results in significantly increased levels of genome editing and transgene expression compared to a single dose. These results demonstrate LNP-mediated ZFN mRNA delivery can drive highly efficient levels of in vivo genome editing and can potentially offer a new treatment modality for a variety of diseases.

Calprotectin is a potent antimicrobial that inhibits the growth of pathogens by tightly binding transition metals such as Mn and Zn, thereby preventing their uptake and utilization by invading microbes. At sites of infection, calprotectin is abundantly released from neutrophils, but calprotectin is also present in non-neutrophil cell types that may be relevant to infections. We show here that in patients infected with the Lyme disease pathogen Borreliella (Borrelia) burgdorferi, calprotectin is produced in neutrophil-free regions of the skin, in both epidermal keratinocytes and in immune cells infiltrating the dermis, including CD68 positive macrophages. In culture, B. burgdorferi's growth is inhibited by calprotectin, but surprisingly, the mechanism does not involve the classical withholding of metal nutrients. B. burgdorferi cells exposed to calprotectin cease growth with no reduction in intracellular Mn and no loss in activity of Mn enzymes including the SodA superoxide dismutase. Additionally, there is no obvious loss in intracellular Zn. Rather than metal depletion, we find that calprotectin inhibits B. burgdorferi growth through a mechanism that requires physical association of calprotectin with the bacteria. By comparison, calprotectin inhibited E. coli growth without physically interacting with the microbe, and calprotectin effectively depleted E. coli of intracellular Mn and Zn. Our studies with B. burgdorferi demonstrate that the antimicrobial capacity of calprotectin is complex and extends well beyond simple withholding of metal micronutrients.

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The study objectiveis to assess effect of the reconstructive stage on the course of the postoperative period in patients with oral cancer.Materials and methods.A retrospective analysis of medical records of 174 patients (121 men and 53 women) aged 36 to 84 years (average 58.26 ± 8.72 years) with oral cancer undergoing treatment from January 2009 to June 2016 was performed. Depending on the nature of the reconstructive stage, the patients were divided into 3 groups. The group 1 consisted of 59 patients, to eliminate the defects of which flaps were taken on axial blood supply, the group 2 included 83 patients who had a reconstructive phase of the operation included a microsurgical reconstruction; 32 patients who had not used additional plastic material to eliminate the defect made up the control group. The following parameters were taken as evaluation parameters: the duration of the operation and hospitalization, the time spent in the intensive care unit, the frequency of serious and frivolous complications. The criterion for distinguishing serious and unserious complications is the fact that the patient returned to the operating room.Results.The reconstructive stage increased the operation duration by 72.12 min in the group 1 and by 285.72 min in the group 2, the length of stay in the intensive care unit – by 0.67 and 2.58 days, respectively, the hospital stay – by 33.9 and 40.4 %. The incidence of complications was higher in the groups 1 and 2 than in the control (6.6 %), and the type of reconstruction had almost no effect on it (42.37 % in the group 1 and 38.55 % in group 2). The frequency of serious complications was higher in the group 2, the frequency of partial flap necrosis – in the group 1.Conclusion.The inclusion of a reconstructive stage into the protocol of surgical treatment for oral cancer is absolutely reasonable, because it gives the patients a chance to return to their normal lives. Microsurgical reconstruction is a costly and labour-consuming procedure; however, it has almost the same number of complication as reconstructive surgery with flaps with an axial blood supply.